Systems, methods and devices for positioning a target

ABSTRACT

Systems, devices, compositions and methods for positioning and/or processing a target are provided. The invention includes systems, devices, methods and related compositions useful, for example, for the separation, isolation, purification, identification, detection and quantification of materials. Also provided are systems and methods for isolation, and/or detection and/or quantification of a target or analyte in a sample. Some systems, devices, compositions and methods comprise oil and aqueous phases stabilized in close proximity to each other. Some systems, devices and methods use a magnetic force to draw a target or carrier-bound through multiple layers. In some embodiments, systems and devices comprise reagents for detection of a target or analyte.

STATEMENT REGARDING RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 63/184,334, filed May 5, 2021, the entire contents of which areincorporated herein by reference for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under R43 OD023021-01A1,awarded by the National Institutes of Health. The government has certainrights in the invention.

INCORPORATION BY REFERENCE

All U.S. patents, U.S. patent applications, publications, foreignpatents, foreign and PCT published applications, articles and otherdocuments, references and publications noted herein, and all thoselisted as References Cited in any patent or patents that issue herefrom,are hereby incorporated by reference in their entirety. The informationincorporated is as much a part of this application as if all the textand other content is repeated in the application and will be treated aspart of the text and content of this application as filed.

TECHNICAL FIELD

The invention generally concerns the separation, isolation,purification, identification, detection and quantification of materials.

Provided herein are systems, devices, compositions and methods forpositioning and/or processing a target. Targets may be positioned in anumber of ways, including positively (by moving or isolating a target,for example, for detection or measurement) and negatively (bypositioning or removing one or more or all non-targets). Using thesystems, devices and methods of the invention, targets or materials towhich targets are attached can be moved, separated, isolated, detected,identified, analyzed, screened for, quantified, or purified using thesystems, devices and methods of the invention. The systems, devices andmethods of the invention include systems, devices and methods for theisolation and/or detection of a target or analyte (including cells,proteins, DNA, RNA or pathogens or parts of pathogens, e.g., proteins,nucleic acids, etc.) in a sample. In particular, provided herein is asystem and a device comprising one or more oil and/or one or moreaqueous phases and/or one or more gas phases stabilized in closeproximity to each other. The systems, devices and methods of theinvention have many uses. For example, they may be used for isolating,separating, moving, purifying, mixing, binding and/or subsequentlydetecting the presence or amount of a target or target analyte from asample or other mixture. Positioning a target may be done by isolating,separating, or moving the target, or by isolating, separating, or movinga material bound to the target with a method, device or system of theinvention, and may be done positively or negatively. In some aspects,provided herein is a system and a device comprising one or morestabilized oil and/or one or more stabilized aqueous phases and/or oneor more gas phases that may be used to move or purify a target oranalyte away from a sample or mixture that contains, may contain, or isor may be suspected of containing the target or analyte using amagnetic, electric, or acceleration-based force (e.g., via gravity orvia a centrifuge) to draw the target or analyte through one or morephases or layers. In some embodiments, the system and the devicecomprises reagents for detection, identification, analysis, isolation orquantification of the target or analyte. The quantification may bepositive-negative for the target, semi-quantitative or quantitative. Theisolation or purification may be complete or partial. One or more or allof the reagents for detection, identification, analysis, isolation orquantification of the target or analyte may be contained in one or moreparts or portions of the system or device, in one or more aqueous and/oroil phases or layers of the system or device, in a base phase or layerof the system or device, in a lower phase, layer or stratum of thesystem or device, or in a terminating or terminal phase, layer orstratum of the system or device (in vertical or latitudinalembodiments), or in a seam, abutment or joint (in horizontal orlongitudinal or other phase/layer orientations in non-vertical ornon-latitudinal embodiments).

BACKGROUND

The following includes information that may be useful in understandingthe present inventions. It is not an admission that any of theinformation is prior art, or relevant, to the presently described orclaimed inventions, or that any publication or document that isspecifically or implicitly referenced is prior art or a reference thatmay be used in evaluating patentability of the described or claimedinventions.

The ability to move, isolate, purify, separate, identify, quantify orotherwise manipulate a target or analyte (e.g., nucleic acid, protein,whole cell, or contaminant) from a complex background is a criticalprerequisite for many common analytical or other processes indiagnostics, biological research, biomarker discovery, forensics, andmore. However, conventional processes, including, for example, analytepurification processes, can be or are time-consuming, expensive, andlaborious, etc., often becoming the bottleneck within such processes,for example, analytical processes. Further, some methodologies damagethe sample or cause undesired loss or inconsistent yield of sample.Accordingly, improved systems, methods and devices for manipulation oftargets, including isolation, separation and purification of a target ortarget analyte, and subsequent rapid detection, identification,quantification of the analyte or other target from a sample are needed,and are provided herein.

BRIEF SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Brief Summary. It is not intended to beall-inclusive, and the inventions described and claimed herein are notlimited to or by the features or embodiments identified in thisintroduction, which is included for purposes of illustration only andnot restriction.

The invention comprises multi-layer systems and devices that provide forautonomous operation of processing steps by the operation of a force toposition a target. The multi-layer systems may be within a container.

In one aspect, the invention provides for autonomous sample preparation(e.g. lysis, washing, and/or solid phase target binding) and testing(e.g., PCR, LAMP, etc.) performed in a single device requiring onlyaddition of a sample, application of a force (e.g. a magnetic force) toobtain and/or read a result. In some embodiments, the inventionoptionally includes a means for communicating a result for viewing,analysis and/or storing (e.g. to a computer or phone).

In some aspects, provided herein are systems, methods and devices andcompositions for isolating or positioning a target from a sample andprocessing the target. In some embodiments, the target of interest isthe target itself. In some embodiments, the target of interest is thetarget bound to a solid phase, or the solid phase itself.

In some embodiments, the system, method or device comprises at least oneaqueous phase or layer and at least one oil phase or layer stabilized inproximity to one another within a container. In some embodiments, the atleast one aqueous phase or layer and the at least one oil phase or layerare stabilized within the container by a hydrophilic porous materialassociated with the at least one aqueous phase and/or a hydrophobicporous material associated with the at least one oil phase. In someembodiments, the at least one aqueous phase and the at least one oilphase are stabilized within the container by modulating geometry or oneor more chemical or physical material characteristics.

In some embodiments where the system, method or device comprises atleast one aqueous phase or layer and at least one oil phase or layer,only the at least one aqueous phase or layer and at least one oil phaseor layer are stabilized. In some embodiments where the system, method ordevice comprises more than one aqueous phase or layer and one or moreoil phases or layers, only one of the aqueous phases or layers isstabilized. In some embodiments where the system, method or devicecomprises more than one aqueous phase or layer and one or more oilphases or layers, more than one or all of the aqueous phases or layersare stabilized. For example, in an embodiment of the invention with fouraqueous phases or layers, one, two, three or all four may be stabilized.In some embodiments where the system, method or device comprises morethan one aqueous phase or layer and one or more oil phases or layers,only one of the oil phases or layers is stabilized. In some embodimentswhere the system, method or device comprises more than one oil phase orlayer and one or more aqueous phases or layers, more than one or all ofthe oil phases or layers are stabilized. For example, in an embodimentof the invention with four oil phases or layers, one, two, three or allfour may be stabilized.

In another embodiment of the invention where the device, system ormethod includes aqueous phases or layers and/or multiple oil phases orlayers, for example, 1-6 aqueous phases or layers and 1-6 oil phases orlayers, from 1-6 of the aqueous phases or layers and/or from 1-6 of theoil phases or layers may be stabilized.

In some embodiments, the device, system or method comprises at least onestabilized aqueous phase or layer. In some embodiments, the at least oneaqueous phase or layer is stabilized by a hydrophilic porous materialassociated with the at least one aqueous phase or layer. In some ofthese embodiments, the device, system or method comprising at least onestabilized aqueous phase or layer does not include an oil phase or layeror a stabilized oil phase or layer. In some embodiments, the device,system or method comprising at least one stabilized aqueous phase orlayer also comprises a gaseous phase or layer. In some embodiments, thegaseous phase or layer comprises, for example, air or an inert gas. Insome embodiments, the gaseous layer comprises helium, neon, argon,krypton, xenon, radon or oganesson, for example. In some of theseembodiments, the device, system or method comprising at least onestabilized aqueous phase or layer includes at least one oil phase orlayer and/or at least one stabilized oil phase or layer. In some ofthese embodiments, the device, system or method comprising at least onestabilized aqueous phase or layer includes at least one oil phase orlayer and/or at least one stabilized oil phase or layer and at least onegaseous layer or phase. In some embodiments, the device, system ormethod comprises at least two stabilized aqueous phases or layers. Insome embodiments, the device, system or method comprising at least onestabilized aqueous phase is within a vessel or container.

In some embodiments, the device, system or method comprises at least onestabilized oil phase or layer. In some embodiments, the at least one oilphase or layer is stabilized by a hydrophobic porous material associatedwith the at least one oil phase or layer. In some of these embodiments,the device, system or method comprising at least one stabilized oilphase or layer does not include an aqueous phase or layer or astabilized aqueous phase or layer. In some embodiments, the device,system or method comprising at least one stabilized oil phase or layeralso comprises a gaseous phase or layer. In some embodiments, thegaseous phase or layer comprises, for example, air or an inert gas. Insome embodiments, the gaseous layer comprises helium, neon, argon,hypton, xenon, radon or oganesson, for example. In some of theseembodiments, the device, system or method comprising at least onestabilized oil phase or layer includes at least one aqueous phase orlayer and/or at least one stabilized aqueous phase or layer. In some ofthese embodiments, the device, system or method comprising at least onestabilized oil phase or layer includes at least one aqueous phase orlayer and/or at least one stabilized aqueous phase or layer and at leastone gaseous layer or phase. In some embodiments, the device, system ormethod comprises at least two stabilized oil phases or layers. In someembodiments, the device, system or method comprising at least onestabilized oil phase is within a vessel or container.

In some embodiments, the least one aqueous phase or layer or the atleast one oil phase or layer is stabilized within a vessel or containerusing a porous material. The material is selected to allow the movementof desired materials through the device or system. The porous materialmay be a mesh. In some embodiments, one or more of the least one aqueousphase or layer is/are stabilized with at least one hydrophilic porousmaterial(s) or mesh(es). In some embodiments, one or more of the leastone oil phase or layer is/are stabilized with at least one hydrophobicporous material(s) or mesh(es). In one embodiment the porous materialand/or the hydrophobic and/or hydrophilic mesh has at least onepredetermined pore size, set of pore sizes or range of pore sizes. Insome embodiments, aqueous and oil phases or layers are stabilized inproximity to one another within a container.

In some embodiments, one or more of the phases or layers are stabilized,and a phase or layer contains fluid with multiple densities and/ordensity gradients.

In some embodiments, the one or more phases or layers may be stabilizedwithin the system or device, e.g. within a container, by modulatingmaterial geometry or one or more chemical or physical materialcharacteristics selected from density, surface chemistry, and porosityof a hydrophilic/hydrophobic porous material, if present in the system

In some embodiments, the systems, devices and methods are designed andused for positioning a target. By way of example, using a system, deviceor method of the invention, one or more targets can be moved, separated,isolated, detected, identified, analyzed, screened for, quantified, orpurified using the systems, devices and methods of the invention. Thesystems, devices and methods of the invention include systems, devicesand methods for the isolation and/or detection of an analyte in asample. In some embodiments, the system, method or device comprises oneor more oil and/or one or more aqueous phases and/or one or more gasphases stabilized in close proximity to each other. These systems anddevices have many uses. For example, they may be used for isolating,separating, moving, purifying, mixing, binding and/or subsequentlydetecting the presence or amount of a target or target analyte from asample or other mixture.

Targets may be positioned positively or negatively, and in a number ofways. Targets may be positioned positively, for example, by isolating atarget (e.g., for detection or measurement). Targets may be positionednegatively, for example, by positioning or removing one or more or allnon-targets.

In some embodiments, positioning a target by, for example, isolating,separating, moving or binding the target or a material bound to thetarget using a method, device or system of the invention may be donepositively.

In some embodiments, positioning a target by, for example, isolating,separating, moving or binding the target or a material bound to thetarget using a method, device or system of the invention may be donenegatively.

In some aspects, provided herein is a system and a device and methodcomprising one or more stabilized oil and/or one or more stabilizedaqueous phases and/or one or more gas phases that may be used to move orpurify a target or analyte away from a sample or mixture that contains,may contain, or is or may be suspected of containing the target oranalyte using a magnetic, electric, or acceleration-based force (e.g.,via gravity or via a centrifuge) to draw the target or analyte throughone or more layers. In some embodiments, the system and the devicecomprises reagents for detection, identification, analysis, isolation orquantification of the target or analyte. The quantification may bepositive-negative for the target, semi-quantitative or quantitative. Theisolation may be complete or partial. One or more or all of the reagentsfor detection, identification, analysis, isolation or quantification ofthe target or analyte may be contained within the system or device.

In some embodiments, the systems, devices, compositions and methods ofthe invention the autonomous operation of processing steps. In someembodiments, the inventions provide for theprocessing/exposure/modification of any solid phase (e.g. para-magneticparticles) that can be moved through the layers/interfaces. In oneaspect, each step functions as a purification/separation step as, by wayof example, the paramagnetic particle passes through a phase, layer orinterface. In other aspects, when the paramagnetic particle, forexample, is within a phase, layer or interface other functionality maytake place (e.g. chemical modification of the solid phase, elution offthe solid phase, etc.). In some embodiments, a solid phase is a solidsupport to which a target has been attached (e.g., fixed, bound,constrained, or sequestered, whether directly or indirectly). However,anything to which a target is attached may serve as a “solid phase.”Semi-solids can serve as solid phases. Solid phases include paramagneticparticles. A mesh or other porous solid support structure used tostabilize a phase or layer of the invention can be a solid phase. Insome embodiments, the target can be the solid phase, e.g. a cell. Inother aspects, each step functions as a purification/separation step as,by way of example, non-target elements of a sample are passed through aphase, layer or interface and the target (attached to a solid, asemi-solid, or a solid phase, e.g. a paramagnetic particle) remainswhile non-target elements are moved away. This is an example of negativeselection.

In some embodiments, the at least one aqueous phase and the at least oneoil phase are stabilized within the container by a hydrophilic porousmaterial associated with the at least one aqueous phase or layer, ahydrophobic porous material associated with the at least one oil phaseor layer, and by modulating surface chemistry or surface energy of aphase or layer such that buoyancy forces of either the one oil phase orlayer or the at least one aqueous phase or layer is overcome and lessthan the surface tension between the at least one oil phase and thehydrophobic porous material or the at least one aqueous phase and thehydrophilic porous material.

In some embodiments, multiple aqueous phases or layers and multiple oilphases and/or gas phases or layers are present in the system. In someembodiments, the system comprises a first aqueous phase or layer, asecond aqueous phase or layer, a first oil phase or layer, and a secondoil phase or layer, with or without one or more gas phases or layers. Insome embodiments, the phases or layers are stacked in an alternatingfashion within the container, such that the first and second aqueousphases or layers are not in direct contact with one another and thefirst and the second oil phases or layers are not in direct contact withone another.

In some embodiments, the container comprises a top opening to permitaddition of a sample to the container. In some embodiments, an aqueousphase is closest to the top opening of the container. In someembodiments, an oil phase is closest to the top opening of thecontainer. In some embodiments, a device or system of the inventionpermits addition of a sample to a device with no top or bottom, e.g., aninsert containing a system of the invention. Phases and/or layers arepositioned as desired in such embodiments and the sample may be added toa layer or phase designated as the “first” or “sample receiving” phaseor layer.

In some embodiments, at least one aqueous phase comprises a lysisbuffer. In some embodiments, at least one aqueous phase comprises a washbuffer.

In some embodiments, the system further comprises paramagnetic particles(PMPs). In some embodiments, the PMPs are housed within the container.The PMPs may be lyophilized or in a liquid form. In some embodiments,the PMPs are housed within the at least one aqueous phase. In someembodiments, the PMPs will bind to a target or target analyte, and maybe referred to as “target-binding” PMPs (or other target capture solidphase). In some embodiments, target-binding PMPs or other target-bindingsolid phases will bind to a target or target analyte, and may bereferred to as “target-binding” PMPs (or other target capture solidphase). In some embodiments, target-binding PMPs or other target-bindingsolid phases are conjugated with a target-binding agent, for example, anantibody, an antibody fragment, a single chain Fv, etc., directed to thetarget and used as a PMP targeting agent. Other useful target-bindingagents include oligonucleotides. In some embodiments, the target-bindingoligonucleotides comprise sequences that target mRNA (e.g., poly dTsequence to bind polyA tails on mRNA) or specific sequences of RNA orDNA.

In some embodiments, provided herein is a system for isolating a targetanalyte from a sample, comprising a first aqueous phase or layer, asecond aqueous phase or layer, a first oil phase or layer, and a secondoil phase or layer. In some embodiments, the phases or layers arestacked in an alternating fashion within a container, such that thefirst and second aqueous phases or layers are not in direct contact withone another and the first and the second oil phases or layers are not indirect contact with one another. In some embodiments, the phases orlayers are stabilized within the container by a hydrophilic porousmaterial associated with the first aqueous phase or layer, a hydrophilicporous material associated with the second aqueous phase or layer, ahydrophobic porous material associated with first oil phase or layer,and a hydrophobic porous material associated with the second oil phaseor layer. In some embodiments, the phases or layers are furtherstabilized within the container by modulating surface chemistry suchthat fluid retention forces associating a fluid layer with a supportstructure dominate other forces (e.g., buoyancy or changes in momentum)that might otherwise disrupt the functional layering or order of thephases.

In some embodiments, the container comprises a top opening to permitaddition of a sample to the container. In some embodiments, the firstaqueous phase or layer is closest to the top opening of the container.In some embodiments, the first oil phase or layer is closest to the topopening of the container. In some embodiments, the first aqueous phaseor layer comprises a lysis buffer. In some embodiments, the secondaqueous phase or layer comprises a wash buffer. In some embodiments, adevice or system of the invention permits the addition of a sample to acontainer or device having no top or bottom as such, e.g., an insertcontaining a system of the invention that does not contain a bottomintegral with its sides. The first and second aqueous phases and thefirst and second oil phases or layers are positioned as desired in suchembodiments and the sample may be added to a layer or phase designatedas a “first” or “sample receiving” phase or layer.

The system may further comprise paramagnetic particles (PMPs). The PMPsmay be housed within the container. In some embodiments, the PMPs arelyophilized. In some embodiments, the PMPs are in a liquid form. In someembodiments, the PMPs are housed within the first aqueous phase.

In any of the embodiments described herein, the system may furthercomprise a magnet. The container may comprise a multi-well plate. Thesystem may further comprise a sample. The sample may be a biologicalsample or a sewage sample. In some embodiments, the biological samplecomprises a nasopharyngeal sample, an oropharyngeal sample, an oral swabsample, an oral sponge sample, a nasal swab sample, a mid-turbinatesample, or a saliva sample.

The systems described herein may be used in methods of isolating anydesired target or material. In some embodiments, the target is nucleicacid. In some embodiments, the target is viral nucleic acid. Forexample, the target may be a SARS-CoV-2 nucleic acid. In someembodiments, the target is a protein (e.g., a hormone or any otherprotein), a carbohydrate, a glycolipid, a cell, a circulating tumorcell, etc. Any material that may be bound to a “solid phase,” asdescribed herein, which in some embodiments may be, e.g., a PMP (eitherattached directly or indirectly) may be a target in one or more of thesystems, devices, compositions and methods of the invention.

One or more or all of the reagents for detection, identification,analysis, isolation or quantification of a target may be contained inone or more parts or portions of the system or device. In someembodiments, one or more or all of the reagents for detection,identification, analysis, isolation or quantification of the target maybe contained in one or more aqueous and/or oil phases or layers of thesystem or device. In some embodiments, the systems and devices describedherein further comprise reagents for detecting the target housed in thebase phase or layer or on a bottom surface of the container. In someembodiments, one or more or all of the reagents for detection,identification, analysis, isolation or quantification of the target arecontained in a lower phase, layer or stratum of the system or device,but above the base layer. In some embodiments, one or more or all of thereagents for detection, identification, analysis, isolation orquantification of the target are contained in a terminating or terminalphase, layer or stratum of the system or device (in vertical orlatitudinal embodiments), or in a seam, abutment or joint (in horizontalor longitudinal or other phase/layer orientations in non-vertical ornon-latitudinal embodiments).

The reagents for detecting the target may comprise reagents for a loopmediated isothermal amplification (LAMP) or a reverse transcriptase loopmediated isothermal amplification (RT-LAMP) assay. In some embodiments,the LAMP or RT-LAMP assay is a colorimetric assay or a fluorescentassay. In other embodiments, the reagents for detecting the targetcomprise reagents for PCR, RT-PCR, qPCR, qtPCR, multiplex PCR, assemblyPCR or asymmetric PCR, for example. In other embodiments, the reagentsfor detecting the target comprise reagents for immunoassays, which mayuse antibodies and/or antibody fragments to detect or measure a targetor target analyte. In some embodiments, the immunoassay is an enzymeimmunoassay, an ELISA (enzyme-linked immunosorbent assay, includingdirect ELISAs, indirect ELISAs, sandwich ELISAs and competitive ELISAs),an IEMA (immunoenzymometric assay), a radioimmunoassay, afluoroimmunoassay, a chemiluminescent immunoassay (CLIA) and countingimmunoassay (CIA). A container housing a PCR reaction, for example, canbe a simple cup shape at the bottom of the device, or in someembodiments, the geometry of the container housing the reaction can besuch that thermocycling is more efficient. For example, in someembodiments, the container housing the reaction can have a high aspectratio to facilitate quicker transfer of heat (i.e., reducing thedistance over which temperature must be conducted to facilitatetemperature cycling of the reaction). In some embodiments, the containerhousing the reaction comprises, consists essentially of, or consists ofa microfluidic channel. In some embodiments, the container housing thereaction is made of different material as the rest of the device. Insome embodiments, the container housing the reaction has integratedheating elements in it.

The devices, systems, methods and compositions of the invention can beused in testing for or assaying for any molecular target, includingbiomolecules, proteins, hormones, nucleic acids, drugs, etc. In someembodiments, devices, systems, methods and compositions of the inventionare used to move, separate, isolate, purify, identify, detect and/orquantify a target, including but not limited to those described orreferred to herein.

In some embodiments, the invention provides a disposable cartridgecomprising a flow-through assay to determine the presence or amount of atarget in a sample comprising a sample application space, a cartridgetop, a cartridge bottom, reagents for detection or quantification of thetarget and an enclosure, the improvement comprising employing with orwithin the enclosure target-binding paramagnetic particles (or othertarget-binding carrier substance), at least one aqueous phase or layerand at least one gaseous or oil phase or layer stabilized in proximityto one another by inclusion of a porous (e.g. target-permeable)structural material associated with the aqueous phase/layer or thegaseous or oil phase/layer or both, and, optionally, a magnet. Otherphases, and/or alternative phases, may be used or included (e.g. two oilphases with or without an aqueous phase or layer).

In some embodiments, the invention provides a flow assay (e.g., lateralflow, vertical flow) device or cartridge comprising a sample applicationportion, a conjugate portion, a test portion and pre-immobilizedreagents in different parts of the device or cartridge, the improvementcomprising employing target-binding paramagnetic particles (or othertarget-binding carrier substance), at least one aqueous phase or layerand at least one gaseous or oil phase or layer stabilized in proximityto one another by inclusion of a porous (e.g. target-permeable)structural material associated with the aqueous phase or the gaseous oroil phase or both, and a magnet. Other phases, and/or alternativephases, may be used or included (e.g. two oil phases with or without anaqueous phase or layer). In some embodiments, the improved flow deviceis designed and/or formatted for use as a disposable, point-of-carecartridge or device.

In some embodiments, the invention provides an immunometric assay todetermine the presence, concentration or amount of a target substance ina sample comprising forming a ternary complex of a first labeled bindingagent, said target substance, and a second binding agent said secondbinding agent being bound to a solid carrier wherein the presence oramount of the substance in the sample is determined by measuring eitherthe amount of labeled binding agent bound to the solid carrier or theamount of unreacted labeled binding agent, the improvement comprisingemploying target-binding solid phase particles (or other target-bindingcarrier substance), at least one aqueous phase or layer and at least onegaseous or oil phase or layer stabilized in proximity to one another byinclusion of a porous structural material associated with the aqueousphase or layer or the gaseous or oil phase or layer or both. Otherphases, and/or alternative phases, may be used or included (e.g. two oilphases with or without an aqueous phase or layer). In some embodiments,the solid phase is a paramagnetic particle and the improved assayincludes or uses a magnet. In some embodiments one or more of thebinding agents is an antibody, an antibody fragment, an oligonucleotide,an aptamer, a peptide, a peptidomimetic, natural or chemically modifiedantisense oligonucleotides, or other suitable agent to assist withcapture of a target. In some embodiments, the immunometric assay ishoused in a single container.

In some embodiments, the invention provides a nucleic acid amplificationtest to determine the presence or amount of a target substance in asample comprising amplifying a nucleic acid sequence and detection ofthe sequence, the improvement comprising employing target-bindingparamagnetic particles (or other target-binding carrier substance), atleast one aqueous phase or layer and at least one gaseous or oil phaseor layer stabilized in proximity to one another by inclusion of a porousstructural material associated with the aqueous phase or layer or thegaseous or oil phase or layer or both, and, optionally, a magnet. Insome embodiments, the nucleic acid amplification test is PCR or RT-PCR.In some embodiments, the nucleic acid amplification test is isothermal.In some embodiments, the isothermal nucleic acid amplification test isreverse transcription polymerase chain reaction (RT-PCR), nickingendonuclease amplification reaction (NEAR), transcription mediatedamplification (TMA), loop-mediated isothermal amplification (LAMP),helicase-dependent amplification (HDA), clustered regularly interspacedshort palindromic repeats (CRISPR), or strand displacement amplification(SDA). In some embodiments, this nucleic acid amplification test ishoused in a single container.

In some embodiments of the invention useful for performing one or moresteps of an assay for the detection or measurement of a target or targetanalyte, one or more of the phases or layers of the device or system maycomprise one or more of several different buffers. In some embodiments,one or more phases or layers comprise a coating buffer, a blockingbuffer, a stabilization buffer, a washing buffer, or act as or comprisea sample diluent. In some embodiments, antibodies or antibody fragmentsare used to generate a detection signal. In some embodiments, the assaycarried out using a device, system or method of the invention comprisesa magnetically-actuated immunoassay in which the movement or positioningof a target or target analyte is achieved using magnetic separationusing a magnetic particle. In some embodiments, the particle used inthese embodiments is made of a core of magnetite that is chemicallymodified by the attachment of antibodies or antibody fragments. In someembodiments, one or more or all components of an assay are used toisolate or purify a target or target analyte.

In some aspects, provided herein are methods for isolating a target froma sample. In some embodiments, provided herein is a method for isolatinga target from a sample comprising adding a sample to a system describedherein, and applying a magnetic force to the system. In someembodiments, the sample is contacted with paramagnetic particles priorto applying the magnetic force to the system. In some embodiments, thePMPs are contacted with the biological sample prior to adding thebiological sample to the system. Contacting the sample with theparamagnetic particles generates one or more target-PMP complexes, andapplying the magnetic force to the system draws the target-PMP complexesthrough phases within the system towards a bottom surface of thecontainer.

In some embodiments, the method further comprises detecting the targetin the biological sample. In some embodiments, the system furthercomprises reagents for detection of the target housed on the bottomsurface of the container, and detecting the target comprises drawing thetarget-PMP complexes through the plurality of porous materials and ontothe reagents for detection of the target. In some embodiments, thereagents comprise reagents for a loop mediated isothermal amplification(LAMP) or a reverse transcriptase loop mediated isothermal amplification(RT-LAMP) assay. In some embodiments, detecting the target comprisesdetecting a signal generated during the LAMP or RT-LAMP assay. In someembodiments, the LAMP or RT-LAMP assay is a colorimetric assay or afluorescent assay.

In some embodiments, the sample is a biological sample or a sewagesample. For example, the biological sample may be a nasopharyngealsample, an oropharyngeal sample, an oral swab sample, an oral spongesample, a nasal swab sample, a mid-turbinate sample, or a saliva sample.In other embodiments, the sample is blood, cerebrospinal fluid, urine,tissue, biopsy tissue, etc. Any type of sample containing or suspectedof containing a target of interest is contemplated for use in thesystems and methods of the invention. In some embodiments, thebiological sample is obtained from a subject suspected of having aninfection. In some embodiments, the subject is suspected of having aviral infection. For example, the subject may be suspected of having aviral upper respiratory infection. In some embodiments, the subject issuspected of having an infection with a SARS-CoV2, a SARS, acoronavirus, a rhinovirus, an influenza virus, or a respiratorysyncytial virus, for example. In some embodiments, the target comprisesviral nucleic acid. For example, the target may comprise a SARS-CoV-2nucleic acid.

In some embodiments, the sample is a sample used to determine paternity.In some embodiments, the sample is for use in prenatal or postnatalscreening.

In some embodiments, the system or device of the invention isbluetooth-enabled, or enabled with another communication functionality(e.g. WiFi, NFC, etc.). In some embodiments, system or device results orresults from a method as described herein are transmitted via bluetoothor other communication functionality to another device (e.g. a phone, atablet, a CPU, a computer, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows one embodiment of a system as described herein. The systemcomprises reagents for LAMP-based detection of the target housed on abottom surface of the container. The system comprises a plurality (2)porous materials. The system comprises a lysis buffer and a wash buffer.As shown in the figure, the system is configured in layers in thefollowing order, from top to bottom: (1) coconut oil, (2) lysis buffer(with associated glass mesh), (3) coconut oil (with associated porous(“porex”) material), (4) wash buffer (with associated glass mesh), (5)coconut oil (with associated porous (“porex”) material), (6) reagentsfor LAMP reaction (with associate glass mesh). Each porous material maybe a hydrophilic glass mesh. Alternatively, one porous material may be aglass mesh and the other porous material may be a synthetic hydrophobicpolymer mesh. The biological sample may be mixed with PMPs, andsubsequently added to the container. A magnet is applied to the bottomof the container, thereby drawing the target-PMP complexes through thelayers and into contact with the LAMP reagents. The container may beincubated at a suitable temperature (e.g. 65° C.) to perform the LAMPassay and subsequently measure the resulting signal. In this embodiment,the resulting signal is colorimetric. In some embodiments the PMPs areconjugated with a target binding agent, for example, an antibody, anantibody fragment, a single chain Fv, etc., directed to the target andused as a PMP targeting agent. Embodiments of the invention can includeas many phases or layers as desired, each phase or layer being with orwithout associated structural materials (including, e.g., materials withdesired porosity), including those structural materials with apreference for a phase or layer.

FIG. 2A shows a side view of one embodiment of a system as describedherein. The container comprises a multi-well plate. One well containsstacked porous materials associated with oil (yellow). In the figure,the porous materials are synthetic hydrophobic polypropylene polymermesh, referred to herein as “porex”, and hydrophilic glass mesh. Thesystem contains 7 mesh materials. From top to bottom, the layers are asfollows: (1) lysis buffer (blue aqueous layer)(stabilized by hydrophilicglass mesh), (2) porex (which stabilizes the oil phase), (3) wash buffer(red aqueous layer)(stabilized by glass mesh), (4) “porex,” (5) washbuffer (blue aqueous layer)(stabilized by hydrophilic glass mesh), (6)“porex,” and (7) LAMP reagents (red aqueous layer)(stabilized byhydrophilic glass mesh).

FIG. 2B shows a bottom view and a top view of the system described inFIG. 1 following application of paramagnetic particles and magnetic pulldown. All three of the systems shown contain oil (yellow), and a glassmesh on the bottom of the well. A comparison of three systems comprising(1) the synthetic polypropylene polymer mesh (referred to as “PorexPad”) associated with oil, (2) no porous material (e.g. only aqueous oroil layers; “+Control”) and a glass mesh and synthetic polypropylenepolymer mesh (“Glass Mesh+Porex Pad”) is shown. As shown in the figure,the synthetic polypropylene polymer mesh (e.g. “porex” pad) and thecombination of the porex pad and glass mesh both permit beads to passthrough the porous material. The “+Control” condition shows what 100%bead transmittance would look like.

FIG. 3 shows a bottom view and a top view following magnetic bead pulldown when small holes were created in the glass mesh material. 1 mmholes allowed for significantly faster pulldown and larger clumps ofbeads. 0.5 mm holes allowed for faster pulldown.

FIG. 4 shows another embodiment of a system as described herein. As inFIG. 1, the system comprises reagents for LAMP-based detection of thetarget housed in a final or terminal layer, e.g., a layer on, or towardor at a bottom surface of the container. In this particular embodiment,the system comprises a Polytetrafluoroethylene (PTFE) O-ring to hold theLAMP reagents on the bottom of the container, and to provide a firmsurface for a porous material to rest on. In this embodiment, the porousmaterial comprises polypropylene (PP) mesh, which is hydrophobic. Thesystem comprises a plurality of porous materials. In this instance, twoporous materials are shown (e.g. two layers). Each porous material maycomprise, consist essentially of, or consist of PP mesh, for example.Alternatively, one material may comprise a PP mesh (hydrophobic) and theother material may comprise a glass mesh (hydrophilic), for example. Thesystem in this embodiment also comprises a lysis buffer and a washbuffer. As shown in the figure, the system is configured in layers inthe following order, from top to bottom: (1) mineral oil, (2) lysisbuffer (hydrophilic glass mesh/fabric), (3) porous material (hydrophobicPP mesh), (4) wash buffer (hydrophilic glass mesh), (5) porous material(hydrophobic PP mesh), (6) PTFE O-ring and reagents for LAMP or otherreaction. The biological sample is mixed with PMPs to which the targetwill bind, and subsequently added to the container. A magnet is appliedto the bottom of the container, thereby drawing the target-PMP complexesthrough the layers and into contact with, for example, the LAMPreagents. The container may be incubated at a suitable temperature (e.g.65° C.) to perform the LAMP assay and subsequently measure the resultingsignal. In this embodiment, the resulting signal is colorimetric.

FIG. 5 shows an image of a system as described in FIG. 4 containing aPTFE O-Ring holding the LAMP reagents on the bottom surface of thecontainer. As shown in the figure, the system labeled “+PP mesh+Glassmesh” is configured in layers in the following order from top to bottom:(1) mineral oil with polypropylene mesh, (2) water with glass mesh, (3)mineral oil with polypropylene mesh, (4) a PTFE O-Ring holding the LAMPreagents. As shown in the figure, the system labeled “+PP mesh” isconfigured in layers in the following order from top to bottom: (1)mineral oil with polypropylene mesh, (2) a PTFE O-Ring holding the LAMPreagents. As shown in the figure, the system labeled “control” isconfigured in layers in the following order from top to bottom: (1) aPTFE O-Ring holding the LAMP reagents. The image is shown after beadpull down, demonstrating that the target-PMP complexes are pulled downinto the center of the O-Ring, thereby contacting the LAMP reagents. Redis shown to indicate where LAMP reagents are located.

FIG. 6 shows another embodiment of a system of the invention. In thisembodiment, the system comprises a custom glycol modified PolyethyleneTerephthalate (PETG) insert to hold the LAMP reagents on the bottom ofthe container. The system comprises a plurality of porous materials. Inthis instance, two porous materials are shown (e.g. two layers). Eachporous material may, for example, comprise a PP mesh. Alternatively, onematerial may comprise a PP mesh and the other material may comprise anylon mesh. The system comprises a lysis buffer and a wash buffer. Asshown in the figure, the system is configured in layers in the followingorder, from top to bottom: (1) mineral oil, (2) an aqueous lysis buffer(stabilized by a hydrophilic nylon mesh), (3) porous material (e.g. a PPmesh) associated with mineral oil, (4) an aqueous wash buffer(stabilized by nylon mesh), (5) porous material (e.g. a PP mesh)associated with mineral oil and (6) reagents for LAMP reaction heldwithin the PETG insert. A sample, e.g. a biological sample, is mixedwith PMPs to which a target, if present in the sample, will bind, andsubsequently added to the container. A magnet is applied to the bottomof the container, thereby drawing target-PMP complexes through thelayers and into contact with the LAMP reagents. The container may beincubated at a suitable temperature (e.g. 65° C.) to perform the LAMPassay and subsequently measure the resulting signal to determine thepresence of amount of the target, if present. In this embodiment, thetarget is present and the resulting signal is colorimetric.

FIG. 7 shows results of a colorimetric LAMP assay following magneticbead pull down using an embodiment of a system as described herein. Abiological sample was mixed with PMPs and added to the system (referredto as a “bead-delivered template”). Data is compared to controls, andcontrols plus a PETG insert. As shown, the system allowed for pull downof sufficient target-PMP complexes (visible as brown spots in thewells), and for successful colorimetric LAMP-based detection of thetarget.

FIG. 8 shows results of a colorimetric LAMP assay to determine the limitof detection (LOD) using primers for SARS-CoV-2

FIG. 9 shows images demonstrating successful target-PMP complexes from asaliva sample. The system comprised a PETG insert. The porous materialscomprised polypropylene and nylon meshes. The saliva sample was dilutedand lysis buffer was added directly to the sample. The sample was heatedto 55° C. for 15 minutes, followed by heating to 98° C. for 3 minutes.The sample was cooled to room temperature, mixed with PMPs, and added tothe container (e.g. added to the wells of a multi-well plate containingthe porous materials and wash buffer). A magnet was applied to thebottom of the container to draw the target-PMP complexes through thepurification layers.

FIG. 10 is a schematic showing an overview of one embodiment of a systemand method of the invention described and claimed herein. A containermay be prepared containing reagents for LAMP-based detection of adesired target housed on a bottom surface of the container. The reagentsmay be secured on the bottom surface by a suitable means, including aninsert (e.g. PETG insert) or an O-Ring. The system comprises a washbuffer and a plurality of porous materials stacked within the container.The system comprises, from top to bottom: (1) polypropylene meshassociated with mineral oil, (2) wash buffer associated with nylon mesh,(3) polypropylene mesh associated with mineral oil, (4) wash bufferassociated with nylon mesh, (5) polypropylene mesh associated withmineral oil, and (6) a PETG insert with LAMP reagents. The container maybe pre-packed into a multi-well plate, wherein each well of the platecontains the contents of a single container. This multi-well plate maybe packaged into a kit. The system further comprises a magnet. In thiscase, the magnet is an array such that each magnet in the array can bealigned with a single well in the multi-well plate. A biological sampleis lysed, mixed with paramagnetic particles, added to the multi-wellplate, and the magnetic array is placed in a suitable position proximalto the bottom of the plate to draw the target-PMP complexes through thepurification layers (e.g. through the porous materials and the washbuffer) and into contact with the LAMP reagents. The plate is incubatedat 65° C., and a signal (e.g. colorimetric, fluorescent, etc., signal)is measured.

FIG. 11A-11C show an embodiment of a system of the invention forisolation and detection of analytes. FIG. 11A shows paramagneticparticles in an aqueous phase (i). Application of magnetic force belowthe system pulls the paramagnetic particles (e.g. the target-PMPcomplexes) through an oil phase (ii, iii, and iv) towards the bottomsurface of the system. FIG. 11B shows a container holding the system.The bottom surface of the container contains reagents for detection ofthe analyte (shown in red). The aqueous and oil phases are stabilized byforces including buoyancy and fluid retention (e.g., wetting, surfacetension, capillary action). FIG. 11C shows an exemplary process forisolating and detecting an analyte using an embodiment of a system ofthe invention described and claimed herein.

FIG. 12 shows a diagram of a point-of-care (POC), single use system asdescribed herein. This embodiment comprises reagents for LAMP-based (orRT-LAMP) detection housed on the bottom surface of the container. Inthis embodiment, the bottom surface of the container includes a septumwhich divides the bottom portion of the container into multiple wellswhich can be filled with reagents for LAMP-based detection, assaying fordifferent portions of the target, i.e. spatially separated multiplexing.In this embodiment, from top to bottom, the system comprises alysis/binding buffer with PMPs (“Extraction Buffer+PMPs”), a solidifiedwax with polypropylene meshes, and LAMP reagents. In this embodiment thesample is a biological sample, containing target or suspected, and issaliva. In this embodiment the sample is added and mixed to thelysis/binding buffer containing PMPs. Upon heating to above the meltingtemperature of the wax, and application of a magnetic field to thebottom of the container, the wax will melt, permitting target-PMPs to bepulled down into contact with the plurality of LAMP reagents. Thecontainer may be incubated at a suitable temperature (e.g. 65° C.) toperform the LAMP assay.

FIG. 13 shows a picture (left) and a cutaway diagram (right) of oneembodiment of a system as described herein. This embodiment of apoint-of-care (POC), single use system comprises reagents for LAMP-based(or RT-LAMP) (green) detection housed on the bottom surface of thecontainer. The system comprises a meltable wax layer (yellow) and aplurality of porous polypropylene materials (“meshes”) (grey). Thesystem comprises a lysis/binding buffer with PMPs (“PMP/SampleMixture”). As shown in the figure, the system is configured in layers inthe following order, from top to bottom: lysis/binding buffer with PMPs,porous material, reagents for LAMP reaction. Each porous material may bea polypropylene mesh, for example. Alternatively, one porous materialmay be a nylon mesh and the other porous material may be a synthetichydrophobic polymer mesh, for example. The sample, e.g. biologicalsample, may be added to the container. A magnet is applied to the bottomof the container, thereby drawing the target-PMP complexes through thelayers and into contact with the LAMP reagents. The container may beincubated at a suitable temperature (e.g. 65° C.) to perform the LAMPassay.

FIG. 14 shows another embodiment of a system as described herein. LikeFIG. 13, in this embodiment, one embodiment of a POC, single-use systemis shown along with a corresponding workflow, from sample acquisition toLAMP reconstitution. In this embodiment, lyophilized LAMP (or RT-LAMP)is housed in the bottom surface of the container in either a bead-formor distributed onto the surface of the bottom of the container. Thesystem comprises a meltable wax layer (yellow), LAMP reconstitutionbuffer, a plurality of porous polypropylene materials (“meshes”)(grey),and lysis/binding buffer with PMPs (“PMP/Sample Mixture”). In someembodiments, the PMPs and the salt components of the lysis/bindingbuffer are frozen into a meltable wax layer, and a sample reconstitutionbuffer is housed separately in the device such that upon melting of thewax, the reconstitution buffer, salt components, and PMPs combine. Insome embodiments, a pierceable membrane is affixed to the top of thecontainer. In this embodiment of a workflow, a biological sample (e.g.saliva, sputum, urine, blood, etc.) is collected in a separate tube. Thetube and container (housing the LAMP reagents and plurality of porousmaterials) are then attached together which pierces the pierceablemembrane which allows for lysis/binding buffer (“Sample Buffer”) andPMPs to mix with the biological sample. Upon inversion and heating ofthe system above the melting temperature of the wax, the LAMP mixtureand LAMP reconstitution buffer will mix.

FIG. 15, is a schematic showing an overview of one embodiment of asystem and method of the invention described and claimed herein. In thisembodiment, the system contains reagents for detecting a target in abiological sample (e.g. saliva, sputum, urine, blood, cell culture mediaetc.) using an enzyme-linked immunosorbent assay (ELISA). The systemcomprises, from top to bottom, a primary antibody binding bufferconsisting in part of antibody-conjugated PMPs with a nylon porousmaterial, mineral oil with a polypropylene porous material, a secondaryconjugate antibody binding buffer (or buffer) comprising a secondaryantibody conjugated to an enzyme (e.g. horseradish peroxidase), asolidified wax layer with a polypropylene porous material, and asubstrate solution containing enzyme substrate (e.g.3,3′,5,5′-Tetramethylbenzidine). In this embodiment, a biological sampleis added to the top of the system and mixed with a primary antibodybinding buffer allowing target to bind to primary antibody. A magneticfield is applied to the bottom of the container causing the target-PMPcomplexes to be pulled through the mineral oil layer, and into thesecondary conjugate antibody binding buffer. If the temperature is abovethe freezing temperature of the secondary conjugate antibody bindingbuffer and below the melting temperature of the wax layer, thetarget-PMP complexes will remain in the secondary conjugate antibodybinding buffer until the temperature increases above the wax meltingtemperature. Incubation in this layer allows for secondary conjugateantibodies to bind to target-PMP complexes. In this embodiment, when thetemperature increases above the melting point of the wax, the conjugatedsecondary antibody-target-PMP complexes are pulled into the substratesolution. Once in the substrate solution conjugated secondaryantibody-target-PMP complexes can catalyze the enzymatic reaction on thesubstrate allowing for detection of target.

FIG. 16 is a schematic showing an overview of one embodiment of a systemand method of the invention described and claimed herein. In thisembodiment, the system contains reagents for isolating and detecting acell-based target (e.g. circulating tumor cells (CTCs), neutrophils,t-cells, mesenchymal stem cells, etc.) in a biological sample (e.g.saliva, sputum, urine, blood, cell culture media etc.). In thisembodiment, the cell-based target is CTCs. The system comprises, fromtop to bottom, CTC binding buffer consisting in part ofantibody-conjugated PMPs with a nylon porous material, mineral oil witha polypropylene porous material, a fluorescent antibody binding bufferconsisting in part of an antibody conjugated to a fluorophore (e.g.green fluorescent protein (GFP), red fluorescent protein (RFP), etc.), asolidified wax layer with a polypropylene porous material, and anaqueous solution (e.g. phosphate-buffered saline, etc.). In thisembodiment, a biological sample containing target cells is added to thetop of the system and mixed with the CTC binding buffer allowing targetcells to bind to antibody-PMPs. A magnetic field is applied to thebottom of the container causing the target-PMP complexes to be pulledthrough the mineral oil layer, and into the fluorescent antibody bindingbuffer. If the temperature is above the freezing temperature of thefluorescent antibody binding buffer and below the melting temperature ofthe wax layer, the target-PMP complexes will remain in the fluorescentantibody binding buffer until the temperature increase above the waxmelting temperature. Incubation in this layer allows for fluorescentantibodies to bind to target-PMP complexes. In this embodiment, when thetemperature increases above the melting point of the wax, thefluorescent antibody-target-PMP complexes are pulled into the aqueous.Once in the aqueous solution, target cells can be counted usingfluorescent microscopy.

DEFINITIONS

As used herein, the term “container” means any device, receptacle, orvessel capable of holding a system of the invention and includes anydevice, receptacle, or vessel in which a method of the invention may beperformed. In some embodiments, the container is a cylinder. In someembodiments, the container is portable. Vessels and containers include,for example, any vessel, container, receptacle, holder, carrier,cartridge, bottle, plate(s), well(s) or storage device capable ofholding a described system. In some embodiments, the vessel is aninjection-molded container with labels or raised lettering inscribed atthe time of manufacturing to eliminate the need for some or all externallabeling. In some embodiments, the container is a disposable orsingle-use container. Vessels and containers may be cooled or heated, orcapable of being cooled or heated, by external or built-in or addedinternal means. Vessels and containers may provide for the stability andmaintenance of fluids, including the one or more phases and layers ofthe invention, during manufacture, storage and shipment. Vessels andcontainers may provide for movement of fluids during use of the systemsand methods of the invention. Vessels and containers that can housesystems of the invention or be used to perform methods of the inventioninclude reaction plates and microtiter plates, including 24-well PCRplates, 96-well plates and 384-well plates and other plate formats.Vessels and containers may provide for electrical, optical, mechanicaland liquid interfaces and utilities, including for the use of detectionand quantification reagents. In some embodiments, a container comprisesa top opening to permit addition of a sample to the container. In someembodiments, the first aqueous phase is closest to the top opening ofthe container. In some embodiments, the first oil phase is closest tothe top opening of the container. In some embodiments, the first aqueousphase comprises a lysis buffer. In some embodiments, the second aqueousphase comprises a wash buffer. In some embodiments of a system or deviceof the invention, the container or construct containing the system hasno integral bottom. In some embodiments, the container has only sidesand is open on both ends. In one such embodiment, the container is aninsert, an example of which is shown in FIGS. 11B and 11C. In suchembodiments, the sample may be added to a layer or phase designated as a“first” or “sample receiving” phase or layer. In the example of aninsert shown FIG. 11C, the terminal layer is a porous plastic screen.The terminal layer a device or system of the invention that allows theaddition of a sample to a container or device that does not contain abottom integral with its sides can be a mesh or any porous material tohold the system and allow it to be run as described. In someembodiments, an open end is used to allow removal of target ortarget-binding particles (e.g., PMPs) from the system (e.g., using amagnet). In some embodiments, the target or target-binding particles areremoved into another vessel or container (e.g., a multi-well plate), orinto or onto a detector (e.g., a reader, a blue-tooth enabled reader orinstrument, etc.) that can accept said target or target-bindingparticles, or onto or into a surface or porous material (e.g., a spotcard for drying and transport of sample for later analysis, etc.).

As used herein, the terms “phase” or “layer” are used interchangeablyand refer to a region of a substance (stabilized or unstabilized asdescribed herein) bounded by one or more other substances. Phases orlayers include aqueous layers, oil layers, gaseous layers, emulsionlayers, particle suspension layers, as well as stabilized versions ofsuch layers or other layers used in a system, device or method. Anexample of a phase or layer would be a volume of air surrounded bywater. Although water and air are miscible (per Henry's law), the liquidform of water and gaseous form of air are not generally considered tosubstantially mix. Stabilized phases or layers as described herein arecompositions of matter of the invention. Stabilized phases or layerscomprise devices and systems of the invention, are used in methods ofthe invention.

As used herein, the term “oil” refers to any of numerous substances,usually liquid or semi-solid substances, that do not dissolve in water.The substances are sometimes greasy substances, and sometimes are fromplant, animal, or mineral sources but can also be non-greasy substances.Oils include carbon- and silicone-based polymeric compounds, mineraloils, silicone oils, paraffin waxes, and fluorinated oils, for example.Oils also include mixtures of oils (e.g. waxes with different meltingtemperatures; polymeric oils with different chain lengths; mineral oiland silicone oil; etc.). Oils also include oil-oil emulsions.

As used herein, the term “oil layer” or “oil phase” means a layer in asystem of the invention that compromises oil and is substantiallyhydrophobic and does not substantially mix with an aqueous layer.Suitable oil layers in systems of the invention include, for example,mineral oil, coconut oil, vegetable oil. As noted, other oils includecarbon- and silicone-based polymeric compounds, mineral oils, siliconeoils, paraffin waxes, and fluorinated oils, for example.

As used herein, the term “interface” means a surface forming a commonboundary or transition zone between adjacent regions, bodies,substances, phases or layers. In some embodiments, an interface refersto the point or transition zone at which independent phases or layers inthe systems, devices and methods of the invention are in contact withone another. For example there is a transition zone between water andair due to the miscibility of water with air (i.e., per Henry's law anddiffusion-based mixing), transitioning from liquid water, to airsaturated with water, to air with some non-saturated level of water. Oneexample of a transition zone within a phase or layer is water withdifferent levels of salinity where differences in density allow forregions with different properties with a transition zone between theregions with intermediate levels of salinity.

As used herein, the term “aqueous” means water-based, comprising water,using or having water as an ingredient. In some embodiments, aqueousmediums or regions contain water and other components. Lysis buffers,wash buffers, and the like may comprise aqueous layers as described inembodiments of systems and methods of the invention. In some embodimentsthe water-based medium contains various concentrations (e.g. 1%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 99.9%) of otherwater-soluble substances such as salts or ionic liquids (e.g. ammoniumsulfate, guanidine isothiocyanate, cyanine dyes, tetraethylammonium andtetrabutylammonium, etc.), polar solvents (e.g. ethanol, phenol,methanol, acetonitrile, etc.), acids/bases (e.g. sulfuric acid, aceticacid, sodium hydroxide, etc.), sugars (e.g. sucrose, glucose, mannose,etc.), or polymer (e.g. polyethylene glycol, hyaluronic acid, chitin,collagen I etc.).

In some embodiments, a phase(s), layer(s) or region(s) in devices,systems, methods and compositions of the invention may be an emulsion,e.g. a dispersion of droplets of one liquid in another in which it isnot soluble or miscible.

In some embodiments, a non-oil liquid phase or layer may be used that isnot water-based, e.g., 100% ethanol, phenol, acetonitrile or othercompatible solvents. These may be referred to as a “non-oil/non-aqueous”phase or layer.

An aqueous layer, phase or region is a water-based layer, phase orregion. An aqueous layer comprises water. As used herein, in someembodiments, the term “aqueous layer” or “aqueous phase” means anaqueous region bounded or surrounded by any “non-aqueous” substances,e.g. device plastic, atmosphere/gaseous substance, oil, wax, etc. Insome constructs, an aqueous layer does not need to be homogeneous andcan have transition zones between multiple aqueous regions withdifferent properties in one aqueous layer, e.g., the layer may containtwo aqueous medium regions with different densities.

As used herein, the term “porous” means having pores or other smallspaces that can hold a gas or liquid or allow a target (or non-target)as defined herein (whether bound or unbound to, or part of, a solidphase or other carrier) to pass through, or not pass through, asdesired. Reference to “porous material” or structure, or to a “porousmesh” or “porous layer” means a material comprising void spaces, i.e.,spaces not occupied by the main framework of atoms that make up thestructure of the material. A material through which a target (bound orunbound to a solid phase) to pass through is an example of a porousmaterial. A material through which non-target materials but not a target(bound or unbound to a solid phase) do not pass through is also anexample of a porous material. A porous material or structure, a porousmesh or a porous layer does not need to be constructed of, or consistsof, a single material, i.e., it does not need to be homogeneous. Aporous material or structure, a porous mesh or a porous layer for use insystems, devices, methods and compositions of the invention may comprisedifferent materials, i.e., it may be heterogeneous or inhomogeneous(e.g., in one embodiment, comprising polystyrene and nylon or spatiallyvariable mixtures).

As used herein, the terms “detect”, “detecting”, or “detection” maydescribe either the general act of discovering or discerning, or thespecific observation of a detectably labeled composition. The term“detecting” when used in reference to a target in a sample refers todetecting either the presence or the absence of the target in thesample. In some embodiments, “detecting” a target in a sample refers todetermining that the target is present in the sample. In someembodiments, “detecting” a target in a sample refers to determining thatthe target is not present in the sample or is not present in sufficientquantities to be detected in the sample.

As used herein, the term “biological sample” is used in the broadestsense and is inclusive of many sample types that may be obtained from asubject. Biological samples may be obtained from animals (includinghumans) and encompass fluids (e.g. urine, blood, blood products, sputum,saliva, etc.), solids, tissues (including biopsy tissue, tumor tissues,bone marrow, etc.), and gases. Biological samples include saliva, bloodproducts, such as plasma, serum and the like. In some embodiments, thebiological sample is a nasopharyngeal sample, an oropharyngeal sample,oral swab or sponge sample, a nasal swab sample, a mid-turbinate sample,or a saliva sample. In some embodiments, the biological sample is asaliva sample. The term “saliva sample” as used herein includes, forexample, a sample of saliva collected from a subject. In someembodiments, the biological sample is a nasopharyngeal (NP) sample. A“nasopharyngeal sample” refers to a sample collected from thenasopharyngeal cavity of a subject and includes, for example, a specimencollected using a swab inserted into the nasal cavity or nasopharynx ofa subject. The biological sample may be subjected to variouspretreatment steps prior to performing a method as described herein. Forexample, the biological sample may be frozen, heated, mixed with variousdenaturants (e.g. guanidium thiocyanate), mixed with viscosity reducingreagents (e.g. DTT), mixed with inhibitors of target degradation (e.g.protease inhibitors, RNAse inhibitors, etc.), mixed with variousbuffers, or subjected to other suitable pre-treatment steps. Any of thesubstances added to the biological sample (e.g. denaturants, viscosityreducing reagents, inhibitors of target degradation, buffers, etc.) maybe added to the biological sample or may be present in a storage bufferpresent in a container into which the sample is collected (e.g. presentwithin a storage buffer in a sample collection tube or other collectiondevice or container). In some embodiments, samples contain or aresuspected of containing a microorganism (e.g. a live or attenuatedpathogenic or disease-causing microorganism).

The term “sample” as used herein is used in the broadest sense and isinclusive of many sample types. In some embodiments, the “sample” is a“biological sample”, as described above. In other embodiments, thesample may be an environmental sample, such as a sewage sample which areuseful, for example, for environmental- and wastewater-basedepidemiology. Thus, in some embodiments, a “sample” will refer to aportion of material taken or selected from a larger quantity ofmaterial. In some embodiments, sample refers to any material containingor suspected of containing a target. In some embodiments, the sample isan entire quantity of material, e.g., blood. In some embodiments, thesample is blood, cerebrospinal fluid, urine, tissue, biopsy tissue, etc.Any type of sample containing or suspected of containing a target ofinterest is contemplated for use in the systems and methods of theinvention.

The term “preference,” in the context of two fluids interacting with asubstrate, e.g. a mesh, as used herein can be defined using the contactangle of the interfacing fluids with the substrate. Association of afluid with a solid surface substrate, for example, at an interface isdictated by the surface properties of the substrate, and the chemicalproperties of the two fluids, be it liquid-liquid, liquid-gas, orgas-gas. A contact angle for a fluid-fluid-material combination isroutinely used to quantify the equilibrium of this interaction and canbe impacted by many factors such as temperature, pressure and surfacecharge. The contact angle is the angle between the surface of thesubstrate and the tangent of the fluid interface where the fluidinterface intersects the substrate. The “preferred” fluid, or the fluidwith preference is the fluid whose contact angle is <90°. In someexamples, these preferences are in the nature of hydrophobic andhydrophilic preferences. Some can be categorized based on the contactangle: e.g. (i) superhydrophilic (0°≤θ<10°), (ii) hydrophilic(10°≤θ<90°), (iii) hydrophobic (90°≤θ<150°), and (iv) superhydrophobic(150°<θ≤180°). In general, a superhydrophobic surface shows a watercontact angle higher than 150° and a sliding angle less than 5°. Thesepreferences are relevant also to other embodiments of the invention thatprovide a stabilized interface system and method, and relatedcompositions. Substrates, support materials, meshes and poroussubstrates and porous support materials and meshes may be selected basedon preferred interfaces and associations with one or more fluids. Thisincludes selection based on fluid preference which impacts whether amesh and fluid can be associated to achieve functional performance.Depending on the conditions, nature of the fluid or the material, thepreference of the material for the fluids) may also change, or bechanged (e.g materials with different preferences may be selected fordifferent layers or conditions, such as, for example, PP-water-mineraloil vs. PP-water-silicone oil, PP-water-oil at 20° C. vs. PP-water-oilat 65°). In some embodiments, fluids (e.g., oil added to replace air) orconditions (e.g., temperature) may be changed or swapped out andreplaced during the use of, or as part of, a method of the invention.For example, an aqueous phase or layer may be replaced with an oil phaseor layer, or temperature may be changed for performance or ease of use.

Some substrates with a preference are porous materials. Some substrateswith a preference are porous structural materials. Some substrates witha preference are meshes. Some porous materials, porous structuralmaterials and meshes are hydrophilic. Some porous materials, porousstructural materials and meshes are selected for their degree ofhydrophilicity. Some porous materials, porous structural materials andmeshes are hydrophobic. Some porous materials, porous structuralmaterials and meshes are selected for their hydrophobicity.

The term “stabilized” as used herein in reference to components of asystem or device described herein indicates that the components maintainfunctionality for their intended purpose(s), or remain immiscible withone another over the course of transport, storage, and/or use of thesystem. By “immiscible” is meant components that do not naturally ortypically naturally or typically form a homogeneous mixture. In general,a stabilized component (e.g. phase or layer) remains functionally apartfrom another component (e.g. another phase or layer). By functionallyapart from is meant a phase or layer that by itself performs, orcontinues to perform, a function in a system, method or device of theinvention. For example, the aqueous and oil phases or layers of a systemdescribed herein are typically immiscible with one another (e.g. thephases or layers remain substantially separated from one another and donot form a homogenous mixture). “Stabilized” may also be used toindicate that this immiscibility of the aqueous and oil layers and/orgas phases or layers remains throughout the life of the system ordevice, or during performance of the method. For example, a supportingstructure used to stabilize a layer may substantially dissolve duringuse (e.g., sucrose initially dried into a mesh structure after wettingwith addition of aqueous medium), altering association of the fluid withthe structure in a desired manner and potentially changing fluidpreference. An interface can be a common boundary between a phase orlayer, a transition zone between a phase or layer, and transition zonewithin a phase or layer, for example. Therefore, “stabilized” regionswithin a phase or layer need not be “immiscible.” The regions within aphase or layer can also be considered stabilized where the componentsform a stable transition zone within a layer or phase.

As used herein, a “stabilized layer or phase” refers to a layer or phase(e.g., an aqueous, oil, or gaseous layer or phase) that is associatedwith a supporting structure that has preference for the fluid of thatlayer or phase relative to at least one other fluid. Preference of thestructure for the associated layer or phase helps to stabilize theassociation of the layer or phase (e.g. fluid) with the supportingstructure and prevent disruption of the phase or layer duringinteractions with other phase(s) or layer(s). As such, other potentiallydisrupting phase(s) or layer(s) or fluid(s) does not have to be part ofthe system of the layers or phases or positioned immediately adjacent tothe layer or phase and may only sometimes come into communication withthe layer or phase (e.g., stabilization might be used as a safetymeasure against unforeseen interactions with fluids not typically in thesystem, or in adding an extraneous material, e.g., a sample, to thesystem. In some embodiments, the supporting structure of a stabilizedlayer is porous in nature, allowing at least some substance, e.g., onemore desired substances, through the structure. The supporting structuredoes not necessarily have to be fixed or bound in orientation orposition but primarily serves to promote association of the preferredfluid to the structure. Further, the supporting structure need not bepermanently associated with a particular phase or layer but may berepositioned for removal from the system or be associated with adifferent phase or layer. A phase or layer can be repositioned, forexample, by manipulating the solid substrate such that it is forcefullyremoved from the phase or layer it was associated with. This may bedone, for example, in order to isolate targets that were negativelyselected for. Likewise, stability afforded by the support structureallows the stabilized phase or layer to be repositioned or reoriented orpassed through other fluids if needed.

As used herein, “associated with” means involved, combined or connectedwith, in whole or in part. The phrase “associated with” includesfunctionally associated with, or connected with. In some embodiments,for example, a substrate, solid phase or structural material (e.g. amesh) is associated with a phase or layer if—wherever a substrate, solidphase or structural material in located or positioned within a system ordevice of the invention in relation to a phase or layer—it provides astabilizing function to the phase or layer. A substrate, solid phase orstructural material need not be immersed within a phase or layer to beassociated with or provide stabilizing function to the phase or layer.

As used herein, the term “immersed” means under the surface of a liquid,in whole or in part. Thus, for example, a mesh may be wholly submergedor within in a liquid phase or layer, or it may be at, near, or on thesurface of a phase or layer, or may be only partly but not wholly withinit. A phase or layer may comprise or consist essentially of a poroussubstrate, solid phase or structural material. A phase or layer may be aporous substrate, solid phase or structural material comprising orconsisting essentially of an oil phase or layer, or the oil layer orphase may comprise or consist essentially of the porous substrate, solidphase or structural material. In some embodiments, for example, a mesh(one example of a porous substrate, solid phase or structural material)can be wholly or partly in oil, or the oil phase or layer may compriseor consist essentially of oil within a mesh. In some embodiments, forexample, a mesh (or other porous substrate, solid phase or structuralmaterial) can be wholly or partly in an aqueous liquid phase or layer,or the aqueous phase or layer may comprise an aqueous liquid within amesh.

The term “stack” or “stacked” as used herein refers to substances (e.g.aqueous phase or layer, oil phase or layer, gaseous phase or layer,porous materials, hydrophobic mesh, hydrophilic mesh, etc.) within thesystem disclosed herein that are aligned (or not aligned) with eachother axially along an axis, for example, the Y-axis (e.g. in a verticalfashion) within a container or the X-axis (e.g. in a horizontal fashion)within a container. The layers may be in any desired 3D orientation. Thelayers do not have to be planar, and the arrangements can be as desired.For example, the system may comprise a plurality of porous materialsthat are “stacked” within the container. The term does not necessarilyindicate that the porous materials are in direct contact with each otherwithin the stack. Rather, the porous materials may be spaced apart or indirect contact in some areas and spaced apart in other areas. Porousmaterials may be associated with or separated by an aqueous phase orlayer (e.g. wash buffer, lysis buffer) and/or associated with orseparated by an oil phase or layer (e.g. mineral oil, coconut oil).

The term “subject” as used herein refers to an entity from which abiological sample is obtained. The subject may be a mammal. In someembodiments, the subject is a human. In some embodiments, the subject isnot a mammal, but an inanimate object. In some embodiments, the subjectis the environment.

The term “target” is used in the broadest sense and refers to anydesired material, including any material within a sample. Targetsinclude, in one embodiment, any material that may bind a paramagneticparticle or other solid phase—either directly, or indirectly, forexample, via a conjugated antibody or antibody fragment—and be pulledfrom a sample by application of a magnetic force. In some embodiments,the target is a protein (e.g. antibody, hormone, etc.), carbohydrate(e.g. glycogen, chitin, etc.), whole cell, cellular component (e.g.mitochondria, exosome, nucleus, etc.), or a nucleic acid (e.g. DNA,RNA). In some embodiments, the target is a metabolite, a carbohydrate, aglycopeptide, or a lipid. Targets include analytes. Targets can alsoinclude material that is not of interest and is instead being removed toenrich for material of interest (e.g., in negative selection). In someembodiments, targets are substances that are moved, separated, isolated,detected, identified, analyzed, screened for, quantified, or purified,for example.

The terms “analyte” or “target analyte” refer to any substance beingidentified or measured.

A “magnet” for use in a system, device or method of the invention refersto a means for generating magnetic force. As used herein, magnetsinclude permanent magnets, temporary magnets and electromagnets.

DETAILED DESCRIPTION

The invention comprises multi-layer and multi-phase systems within acontainer that provide for autonomous operation of processing steps bythe operation of a force to position a target.

In one aspect, the invention provides a self-contained system and devicefor sample preparation and target testing (e.g., PCR, LAMP, etc.)performed in a single container requiring only addition of a sample,application of a force (e.g. a magnetic force) and, in some embodiments,reading a result.

In some aspects, provided herein are systems, methods and devices forisolating or positioning a target from a sample and processing thetarget. In some embodiments, the target of interest is the targetitself. In some embodiments, the target of interest is the target isbound to a solid phase or is the solid phase itself. In someembodiments, the target is isolated and detected. In some embodiments,the target is purified. In some embodiments, the target is quantified.In some aspects, provided herein are systems, devices, compositions andmethods for positioning and/or processing a target. Targets or materialsto which targets are bound may be positioned according to the inventionin a number of ways, including positively (by isolating a target, forexample, or removing a target from a sample, for detection ormeasurement or disposal) and negatively (by positioning or removing oneor more or all non-targets). Using the systems, devices and methods ofthe invention, targets can be moved, separated, isolated, detected,identified, analyzed, screened for, quantified, or purified using thesystems, devices and methods of the invention. The systems, devices andmethods of the invention include systems, devices and methods for theisolation and/or detection of a target or analyte (including pathogensor parts of pathogens, e.g., proteins, nucleic acids, etc.) in a sample.In particular, provided herein is a system and a device comprising oneor more oil and/or one or more aqueous phases and/or one or more gasphases stabilized in close proximity to each other. The systems, devicesand methods of the invention have many uses. For example, they may beused for isolating, separating, moving, purifying, mixing, bindingand/or subsequently detecting the presence or amount of a target ortarget analyte from a sample or other mixture.

In some aspects, provided herein is a system and a device comprising oneor more stabilized oil and/or one or more stabilized aqueous phasesand/or one or more gas phases that may be used to move or purify atarget or analyte away from a sample or mixture that contains, maycontain, or is or may be suspected of containing the target or analyteusing a force. Forces include any force, including magnetic, electric,convective, or acceleration-based force (e.g., via gravity or via acentrifuge), for example, to draw the target or analyte through one ormore phases or layers.

In some embodiments, the system and the device comprises reagents fordetection, identification, analysis, isolation or quantification of thetarget or analyte. The quantification may be positive-negative for thetarget, semi-quantitative or quantitative. The isolation or purificationmay be complete or partial. One or more or all of the reagents fordetection, identification, analysis, isolation or quantification of thetarget or analyte may be contained in one or more parts or portions ofthe system or device, in one or more aqueous and/or oil phases or layersof the system or device, for example, in a base phase or layer of thesystem or device. In some embodiments, one or more or all of thereagents for detection, identification, analysis, isolation orquantification of the target are contained in a lower phase, layer orstratum of the system or device, but above the base layer. In someembodiments, one or more or all of the reagents are in a lower phase,layer or stratum of the system or device, or in a terminating orterminal phase, layer or stratum of the system or device (in vertical orlatitudinal embodiments). In some embodiments, one or more or all of thereagents in a seam, abutment or joint (in horizontal or longitudinal orother phase/layer orientations in non-vertical or non-latitudinalembodiments). In some embodiments, one or more or all of the reagentsfor detection, identification, analysis, isolation or quantification ofthe target are contained in a terminating or terminal phase, layer orstratum of the system or device (in vertical or latitudinalembodiments), or in a seam, abutment or joint (in horizontal orlongitudinal or other phase/layer orientations in non-vertical ornon-latitudinal embodiments). These are examples of reagent placement,but do not include all possible placements, which will be as desired oras appropriate in light of the conformation of the system or device, orthe desired performance.

The systems described herein may be used in methods of isolating anydesired target or material. In some embodiments, the target is nucleicacid. In some embodiments, the target is viral nucleic acid. Forexample, the target may be s SARS-CoV-2 nucleic acid. In someembodiments, the target is a protein (e.g., a hormone or any otherprotein), a carbohydrate, a glycolipid, a cell, a circulating tumorcell, etc. Any material that may be bound to a PMP (either directly orindirectly) may be a target in one or more of the systems, devices,compositions and methods of the invention.

In some embodiments where the system, method or device comprises atleast one aqueous phase or layer and at least one oil phase or layer,only the at least one aqueous phase or layer and at least one oil phaseor layer are stabilized.

In some embodiments where the system, method or device comprises morethan one aqueous phase or layer and one or more oil phases or layers,only one of the aqueous phases or layers is stabilized. In someembodiments where the system, method or device comprises more than oneaqueous phase or layer and one or more oil phases or layers, more thanone or all of the aqueous phases or layers are stabilized. For example,in an embodiment of the invention with four aqueous phases or layers,one, two, three or all four may be stabilized. In some embodiments wherethe system, method or device comprises more than one aqueous phase orlayer and one or more oil phases or layers, only one of the oil phasesor layers is stabilized. In some embodiments where the system, method ordevice comprises more than one oil phase or layer and one or moreaqueous phases or layers, more than one or all of the oil phases orlayers are stabilized. For example, in an embodiment of the inventionwith four oil phases or layers, one, two, three or all four may bestabilized.

In another embodiment of the invention where the device, system ormethod includes aqueous phases or layers and/or multiple oil phases orlayers, for example, 1-6 aqueous phases or layers and 1-6 oil phases orlayers, from 1-6 of the aqueous phases or layers and/or from 1-6 of theoil phases or layers may be stabilized.

In some embodiments, the device, system or method comprises at least onestabilized aqueous phase or layer. In some embodiments, the at least oneaqueous phase or layer is stabilized by a hydrophilic porous materialimmersed, on, in, or otherwise associated with the at least one aqueousphase or layer. In some of these embodiments, the device, system ormethod comprising at least one stabilized aqueous phase or layer doesnot include an oil phase or layer or a stabilized oil phase or layer. Insome embodiments, the device, system or method comprising at least onestabilized aqueous phase or layer also comprises a gaseous phase orlayer. In some embodiments, the gaseous phase or layer comprises, forexample, air or an inert gas. In some embodiments, the gaseous layercomprises helium, neon, argon, krypton, xenon, radon or oganesson, forexample. A gas may be a mixture of gases (e.g., air; air with volatiles;helium and neon, etc.). A gas may be in plasma form. In some of theseembodiments, the device, system or method comprising at least onestabilized aqueous phase or layer includes at least one oil phase orlayer and/or at least one stabilized oil phase or layer. In some ofthese embodiments, the device, system or method comprising at least onestabilized aqueous phase or layer includes at least one oil phase orlayer and/or at least one stabilized oil phase or layer and at least onegaseous layer or phase. In some embodiments, the device, system ormethod comprises at least two stabilized aqueous phases or layers. Insome embodiments, the device, system or method comprising at least onestabilized aqueous phase is within a vessel or container. In someembodiments, the device, system or method comprises at least onestabilized aqueous phase or layer and at least one gaseous phase orlayer but not an oil phase or layer.

In some embodiments, the device, system or method comprises at least onestabilized oil phase or layer. In some embodiments, the at least one oilphase or layer is stabilized by a hydrophobic porous material associatedwith the at least one oil phase or layer. In some of these embodiments,the device, system or method comprising at least one stabilized oilphase or layer does not include an aqueous phase or layer or astabilized aqueous phase or layer. In some embodiments, the device,system or method comprising at least one stabilized oil phase or layeralso comprises a gaseous phase or layer. In some embodiments, thegaseous phase or layer comprises, for example, air or an inert gas. Insome embodiments, the gaseous layer comprises helium, neon, argon,krypton, xenon, radon or oganesson, for example. In some of theseembodiments, the device, system or method comprising at least onestabilized oil phase or layer includes at least one aqueous phase orlayer and/or at least one stabilized aqueous phase or layer. In some ofthese embodiments, the device, system or method comprising at least onestabilized oil phase or layer includes at least one aqueous phase orlayer and/or at least one stabilized aqueous phase or layer and at leastone gaseous layer or phase. In some embodiments, the device, system ormethod comprises at least two stabilized oil phases or layers. In someembodiments, the device, system or method comprising at least onestabilized oil phase is within a vessel or container. In someembodiments, the device, system or method comprises at least onestabilized oil phase or layer and at least one gaseous phase or layerbut not an aqueous phase or layer.

In some embodiments, the least one aqueous phase or layer or the atleast one oil phase or layer is stabilized within a vessel or containerusing a porous material. The material is selected to allow the movementof desired materials through the device or system. The porous materialmay be a mesh. In some embodiments, one or more of the least one aqueousphase or layer is/are stabilized with at least one hydrophilic porousmaterial(s) or mesh(es). In some embodiments, one or more of the leastone oil phase or layer is/are stabilized with at least one hydrophobicporous material(s) or mesh(es). In one embodiment the porous materialand/or the hydrophobic and/or hydrophilic mesh has at least onepredetermined pore size, set of pore sizes or range of pore sizes. Insome embodiments, aqueous and oil phases or layers are stabilized inproximity to one another within a container.

In some embodiments, the one or more phases or layers may be stabilizedwithin the container by modulating material geometry or one or morechemical or physical material characteristics selected from density,surface chemistry, and porosity of a hydrophilic/hydrophobic porousmaterial, if present in the system.

In some embodiments, the systems, devices and methods are designed andused for positioning a target. By way of example, using a system, deviceor method of the invention, one or more targets can be moved, separated,isolated, detected, identified, analyzed, screened for, quantified, orpurified using the systems, devices and methods of the invention. Thesystems, devices and methods of the invention include systems, devicesand methods for the isolation and/or detection of an analyte in asample. In some embodiments, the system, method or device comprises oneor more oil and/or one or more aqueous phases and/or one or more gasphases stabilized in close proximity to each other. These systems anddevices have many uses. For example, they may be used for isolating,separating, moving, purifying, mixing, binding and/or subsequentlydetecting the presence or amount of a target or target analyte from asample or other mixture.

Targets may be positioned positively or negatively, and in a number ofways. Targets may be positioned positively, for example, by moving orisolating a target or removing a target from a sample (e.g., fordetection or measurement, or disposal, etc.). Targets may be positionednegatively, for example, by positioning or removing one or more or allnon-targets.

In some embodiments, positioning a target by, for example, isolating,separating, moving or binding the target or a material bound to thetarget using a method, device or system of the invention may be donepositively.

In some embodiments, positioning a target by, for example, isolating,separating, moving or binding the target or a material bound to thetarget using a method, device or system of the invention may be donenegatively.

In some aspects, provided herein is a system and a device and methodcomprising one or more stabilized oil and/or one or more stabilizedaqueous phases and/or one or more gas phases that may be used to move orpurify a target or analyte away from a sample or mixture that contains,may contain, or is or may be suspected of containing the target oranalyte using a magnetic, electric, or acceleration-based force (e.g.,via gravity or via a centrifuge) to draw the target or analyte throughone or more layers. In some embodiments, the system and the devicecomprises reagents for detection, identification, analysis, isolation orquantification of the target or analyte. The quantification may bepositive-negative for the target, semi-quantitative or quantitative. Theisolation may be complete or partial. One or more or all of the reagentsfor detection, identification, analysis, isolation or quantification ofthe target or analyte may be contained the system or device.

In some embodiments, the systems, devices, compositions and methods ofthe invention the autonomous operation of processing steps. In someembodiments, the inventions provide for theprocessing/exposure/modification of any solid phase (e.g. para-magneticparticles) that can be moved through the layers/interfaces. In oneaspect, each step functions as a purification/separation step as, by wayof example, the paramagnetic particle passes through a phase, layer orinterface. In other aspects, when the paramagnetic particle, forexample, is within a phase, layer or interface other functionality maytake place (e.g. chemical modification of the solid phase, elution offthe solid phase, etc.). As noted, a solid phase is a solid support towhich a target has been bound, attached or fixed (whether directly orindirectly). Solid phases include paramagnetic particles. Semi-solidscan serve as solid phases. In some embodiments, anything to which atarget is attached may serve as a “solid phase.” A mesh other poroussolid support structure is not generally considered a solid phase. Insome embodiments, the target can be the solid phase, e.g. a cell.

One embodiment of the invention is the application of a system, deviceor method as described herein to a specific target or analyte in aspecific matrix.

In some aspects, provided herein are systems and methods for isolating atarget from a sample. In some embodiments, the systems and methods ofthe invention are used for detecting and/or quantifying a target in asample. In some embodiments, the systems and methods of the inventionare used for determining the presence or amount of a target in a sample.In some embodiments, the systems comprise a container housing at leastone aqueous phase (e.g. aqueous layer) and at least one oil phase (e.g.oil layer). The aqueous and oil phases or layers are stabilized in thecontainer and, in some embodiments, are stabilized in close or otherwisefunctional proximity to each other within the container, the distancebetween being as desired or needed for the function of the system,method or device.

The system may comprise any suitable or desired number of aqueous andoil and/or gaseous phases or layers to facilitate isolation of thetarget analyte. In some embodiments, the system comprises one aqueousphase. In some embodiments, the system comprises more than one aqueousphase. In some embodiments, the system comprises one oil phase. In someembodiments, the system comprises more than one oil phase. In someembodiments, the system comprises one aqueous phase and one oil phase.In some embodiments, the system comprises more than one aqueous phaseand more than one oil phase. In some embodiments, the system comprisesat least two aqueous phases and at least two oil phases. In someembodiments, the aqueous and oil phases are stacked in an alternatingfashion, such that no two aqueous phases are in direct contact with eachother and no two oil phases are in direct contact with each other. Insome embodiments, the phase closest to the top of the container (e.g.the phase that will contact the sample first) is an aqueous phase. Inother embodiments, the phase closest to the top of the container is anoil phase. For example, in some embodiments the aqueous and oil phasesare stacked in an alternating fashion, such that the system comprises,from top to bottom, a first aqueous phase, a first oil phase, a secondaqueous phase, and a second oil phase. In other embodiments, the systemcomprises, from top to bottom, a first oil phase, a first aqueous phase,a second oil phase, and a second aqueous phase. In some embodiments, thesystem comprises at least three aqueous phases and at least three oilphases, at least four aqueous phases and at least four oil phases, atleast five aqueous phases and at least five oil phases, etc. In someembodiments, one or more of the aqueous phases are stabilized. In someembodiments, one or more of the oil phases are stabilized. In someembodiments, one or more of the aqueous and one or more of the oilphases are stabilized.

In some embodiments, at least one aqueous phase comprises a lysisbuffer. In some embodiments, the lysis buffer is the first aqueous phase(e.g. the aqueous phase closest to the top of the container). A suitablelysis buffer is chosen based on the nature of the sample and the target.Accordingly, the sample may be added to the system, such that the samplecontacts the lysis buffer prior to coming into contact with any othercomponents of the system. For example, the lysis buffer may be housedwithin the container above the plurality of porous materials and aboveany oil phases present in the container, such that the biological samplecontacts the lysis buffer prior to passing through the plurality ofporous materials. This facilitates lysis of cells contained within thesample, thereby facilitating release of the target analyte containedtherein prior to isolation, purification, or evaluation of the presenceor amount of the target. In other embodiments, the first aqueous phaseis below the first oil phase (e.g. the first oil phase is closest to thetop of the container). In such embodiments, the first oil phase willhelp to remove potential contaminants from the sample prior to lysingthe sample to release the target analyte.

In some embodiments, a lysis buffer may be housed in between one or morelayers of the plurality of porous materials (e.g. in between one porousmaterial and another). In some embodiments, the lysis buffer is housedabove the plurality of porous materials and/or in between one or morelayers of the plurality of porous materials. In other words, a firstaqueous phase and a second aqueous phase may comprise a lysis buffer. Insome embodiments, for example, in nucleic acid isolation, a first lysisstep is performed followed by a wash step with the lysis or otherbuffer. In some embodiments, the lysis buffer is added to the biologicalsample prior to adding the sample to the system. For example, a lysisbuffer may be added to the biological sample as part of one or morepre-treatment steps performed prior to adding the sample to the system.

Any suitable lysis buffer may be used. In some embodiments, the lysisbuffer comprises a salt (e.g. NaCl, KCl, (NH₄)₂SO₄, etc.). In someembodiments, the lysis buffer comprises a detergent. For example, thebiological sample may comprise an ionic detergent (e.g. sodium dodecylsulfate, deoxycholate, cholate, etc.), a non-ionic detergent (e.g.Triton X-100, DDM, digitonin, Tween 20, Tween 40, NP-40, PluronicF-127), a zwitterionic detergent, or a chaotropic detergent. In someembodiments, lysis buffer comprises 0-5% detergent (v/v). For example,the biological sample may comprise 0%, about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%,about 4%, about 4.5%, or about 5% detergent. Detergents are most widelyused for lysing mammalian cells. For lysing bacterial cells, the cellwall has to be broken down in order to access the cell membrane, anddetergents are often used along with lysozymes. Agents for lysingviruses for downstream assays are virus dependent, and known in the art.The lysis buffer may be brought to a suitable volume for subsequent useby the addition of a suitable buffer. For example, the lysis may bebrought to a suitable volume by the addition of phosphate bufferedsaline (PBS), Tris hydrochloride, saline, and the like. The lysis buffermay comprise one or more enzymes or chemical agents to assist withbreaking down the contents therein to facilitate release of the desiredtarget. For example, the lysis buffer may further comprise one or moreenzymes, such as one or more proteases. In particular embodiments, thelysis buffer may comprise proteinase K. The lysis buffer mayadditionally comprise one or more suitable reagents to preventdegradation of the target within the sample. For example, suitablereagents and/or inhibitors (e.g. RNase inhibitors, nuclease inhibitors,etc.) may be added to the lysis buffer prior to use in a system asdescribed herein.

In some embodiments, at least one aqueous phase comprises a wash buffer.The purpose of the wash buffer is generally to dilute unwantedcomponents of a sample that are carried into the wash buffer layer by,for example, by PMPs, where PMPs are used, prior to moving the PMPs intothe next phase or layer. Another purpose is to promote desorption ofunwanted sample components bound to the PMPs, for example, prior tomoving them into the next layer. A wash or wash buffer may also be usedto “mitigate” (e.g., chemically) an unwanted sample component carriedinto the wash layer prior to moving it to the next layer.

In some embodiments, the aqueous phase comprising the wash buffer is notthe first aqueous phase (e.g. is not the aqueous phase closest to thetop of the container). For example, the wash buffer may be the secondaqueous phase, the third aqueous phase, the fourth aqueous phase, etc.In some embodiments, multiple aqueous phases comprise a wash buffer. Forexample, the first aqueous phase may comprise a lysis buffer, and thesecond and third aqueous phases may comprise a wash buffer. In someembodiments, the aqueous phases comprising the wash buffer reside belowthe aqueous phase comprising the lysis buffer, and above the reagentsfor detecting the target. In some embodiments, the wash buffer compriseswater. In some embodiments, the wash buffer comprises ethanol. In someembodiments, the wash step or wash buffer is performed with a lysisbuffer or a mixture of wash and lysis buffers.

In some embodiments, the system further comprises paramagneticparticles. In some embodiments, one or more aqueous phases furthercomprises paramagnetic particles (PMPs). In some embodiments, the firstaqueous phase further comprises paramagnetic particles. The paramagneticparticles bind to the target analyte, thus creating one or moretarget-PMP complexes. In some embodiments, PMPs bind to a target ortarget analyte, and are referred to as “target-binding” PMPs (or othertarget capture solid phase). In some embodiments, target-binding PMPs orother target-binding solid phases are conjugated with a target-bindingagent, for example, an antibody, an antibody fragment, a single chainFv, oligonucleotide, aptamer, peptidomimetic, etc., directed to thetarget and used as a PMP targeting agent, “target-binding” PMP, asdescribed. Any suitable paramagnetic particle may be used. In someembodiments, paramagnetic particles may be purchased from a commercialvendor. The specific type of paramagnetic particle used depends on thetarget to be isolated from the sample. For example, particles with arelatively large surface area may be preferable for binding nucleicacid, such as viral RNA. In some embodiments, as noted, one or moreparamagnetic particles may be functionalized to aid incapture/purification of the target. For example, some or all of theparamagnetic particles may be functionalized with one or moreantibodies, antigen-binding fragments (e.g., F(ab′)2, Fab, Fab′, Fv,etc., generated form the variable region of IgG and IgM, for example,which may vary in size, valency and Fc content), single chain variablefragments (scFV) recombinant antibody fragments (rAbFs), aptamers,peptides and peptidomimetics, natural and chemically modified antisenseoligonucleotides, or other suitable agents to assist with capture of atarget. In some embodiments, different paramagnetic particles arefunctionalized for different targets such that one group of paramagneticparticles can function to indicate successful interaction with and/orisolation from a sample (e.g., a particle targeting human RNaseP RNA/DNAin saliva as a means to indicate sample was successfully lysed and orthat PMPs and sample were successfully mixed and subsequently isolated).In some embodiments, different sets of paramagnetic particles can serveas positive or negative controls. In some embodiments, the paramagneticparticles may be functionalized with one or more spike proteinantibodies to assist with the capture of SARS, coronavirus, SARS-CoV-2and related targets. As used herein, reference to paramagnetic particlesor PMPs includes functionalized paramagnetic particles.

The paramagnetic particles may be lyophilized or dried. PMPs may becontained in a lyophilized or dried mixture or solution. In otherembodiments, the paramagnetic particles may be in a liquid formulation.The paramagnetic particles are contacted with the sample, thusgenerating a plurality of target-PMP complexes. In some embodiments, theparamagnetic particles are housed within the container holding theplurality of porous materials. For example, the PMPs may be a part of afirst aqueous phase. Alternatively, the paramagnetic particles may behoused separately (e.g. in a separate container from the plurality ofporous materials). When housed separately, the paramagnetic particlesmay be added to the container housing the plurality of porous materialsprior to adding the sample to the container, after adding the sample tothe container, or concurrently with adding the sample to the container.For example, the PMPs may be added to a first aqueous phase present inthe container. In some embodiments, the first aqueous phase alsocomprises a lysis buffer, such that addition of the sample to thecontainer results in lysis of cells contained therein and binding of thetarget or analyte to the PMPs present within the aqueous layer.Alternatively, the paramagnetic particles may be mixed with the sampleto generate a composition comprising a plurality of target-PMPcomplexes, and the composition may be added to the container.

Any suitable amount of PMPs may be contacted with the sample. Inembodiments where the PMPs are contained in a liquid formulation, anysuitable volume of the liquid composition comprising paramagneticparticles may be contacted with the sample. In some embodiments, thevolume of the liquid composition comprising the PMPs may equal or exceedthe volume of the sample. For example, the volume of the liquidcomposition comprising PMPs may be at least 100%, at least 150%, atleast 200%, at least 250%, at least 300%, at least 350%, at least 400%,at least 450%, or at least 500% the volume of the sample.

Any suitable concentration of PMPs may be used to ensure sufficientbinding of the PMPs to the target (e.g. formation of a sufficient numberof target-PMP complexes). For lyophilized PMP formulations, any suitableweight of lyophilized product may be used to ensure the properconcentration of PMPs to be contacted with the sample. For liquidformulations, the liquid composition comprising the PMPs may compriseany suitable concentration of PMPs to ensure sufficient binding of thePMPs to the target (e.g. formation of a sufficient number of target-PMPcomplexes). For example, PMPs may be present in the liquid compositionat about 1-20% (v/v). For example, PMPs may be present in the liquidcomposition in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or about 20% (v/v).

In some embodiments, the liquid composition comprising PMPs containsother suitable reagents for processing/handling of samples. For example,the liquid composition comprising PMPs may contain one or moredetergents, reducing agents, buffers, inhibitors, enzymes (e.g.proteases), denaturants, etc. Any additional reagents present in thesample may additionally be present in the liquid composition comprisingPMPs. For example, the liquid composition may further comprise one ormore reagents to decrease viscosity of the sample. For example, theliquid composition may comprise PMPs and DTT. The liquid composition maycomprise other suitable buffers, inhibitors, and the like to preventdegradation of the target (e.g. target nucleic acid, target protein,etc.) during sample processing. Suitable inhibitors that may be presentin the liquid composition comprising PMPs include, for example, RNaseinhibitors, protease inhibitors, nuclease inhibitors, and the like.Lyophilized PMP formulations may contain other suitable reagentscommonly used in the lyophilization process, including bulking agents,stabilizers, and other suitable excipients.

In some embodiments, the systems described herein further comprise atleast one oil phase. The oil phase may be any suitable hydrophobicliquid. In some embodiments, the oil phase may comprise mineral oil,coconut oil, vegetable oil, and the like. In some embodiments, an oilphase (e.g. a layer of light mineral oil, coconut oil, etc.) residesabove the plurality of porous materials and above the wash buffer and/orlysis buffer, if present in the system. Accordingly, the sample willpass through the layer of oil prior to contacting the lysis buffer.

The aqueous and oil phases may be stabilized within the container by oneor more factors. In some embodiments, the aqueous and oil phases arestabilized, at least in part, by the use of porous materials. Forexample, the systems described herein may comprise a plurality of porousmaterials. The plurality of porous materials are stacked within thecontainer, such that the target (e.g. target-PMP complexes) passesthrough multiple porous layers during the purification process. In someembodiments, the porous materials are not in direct contact with oneanother within the stack. For example, one or more porous materials maybe separated by an aqueous phase (e.g. wash buffer, lysis buffer) or anoil phase (e.g. mineral oil, coconut oil). In some embodiments, one ormore porous materials are in direct contact with one another.

Any suitable porous material may be used. In some embodiments, theporous material is hydrophilic. In some embodiments, the hydrophilicporous material is within or comprises an aqueous phase or layer. Insome embodiments, the porous material is hydrophobic. In someembodiments, the hydrophobic porous material is within or comprises anoil phase or layer. In some embodiments, the porous material is fibrousglass material. For example, the porous material may be a fibrous,hydrophilic glass mesh. In some embodiments, the porous material is asynthetic mesh material. For example, the porous material may comprise apolypropylene mesh, a polyethylene mesh, a polyester mesh, a nylon mesh,or a polyetheretherketone (PEEK) mesh. In some embodiments, thesynthetic mesh material is hydrophobic. In some embodiments, thesynthetic mesh material is hydrophilic. For example, nylon-6 is anexemplary synthetic mesh material that is hydrophilic. Nylon-6 andnylon-6 capillary-channeled polymer (C-CP) fibers are hydrophilic.

In some embodiments, each of the porous materials are the same. In otherwords, the system comprises a plurality of porous materials stackedwithin the container, and each layer in the stack comprises the sameporous material. In other embodiments, one or more of the porousmaterials are different from one or more other porous materials. Inother words, the system comprises a plurality of porous materialsstacked within the container, and one or more layers in the stack isdifferent from another layer.

In various embodiments, the porous material is selected based on thesize of the pores or openings in the material and the size of the targetor analyte, the size of the target or analyte bound to a carrier orsolid phase (e.g. a PMP) and/or the size of elements in the sampledesired to be excluded during the method. In some embodiments, the sizeof the pores or openings in one or more of the porous materials aredifferent from those in one or more other porous materials. In someembodiments, the size of the pores or openings in one or more of theporous materials are the same as those in one or more other porousmaterials in the system.

In some embodiments, a hydrophilic porous material is associated with atleast one aqueous phase or layer, and a hydrophobic porous material isassociated with at least one oil phase or layer. For example,hydrophilic porous material (e.g. glass mesh, nylon) may be associatedwith one or more aqueous phases or layers within the container, and asynthetic hydrophobic mesh material may be associated with at least oneoil phase or layer. In some embodiments, each aqueous phase or layercomprises or consists essentially of a hydrophilic porous material andeach oil phase or layer comprises or consists essentially of ahydrophobic porous material. In some embodiments, the first aqueousphase or layer comprises or consists essentially of a lysis buffer andis stabilized by a hydrophilic porous material (e.g. glass mesh, nylon)associated with the first aqueous phase or layer. In some embodiments,the first aqueous phase or layer and at least one additional aqueousphase or layer comprises or consists essentially of a hydrophilic porousmaterial. For example, the first aqueous phase or layer may comprise orconsists essentially of a lysis buffer, and the second aqueous phase orlayer (and potentially a third aqueous phase or layer, a fourth aqueousphase or layer, etc.) comprises or consists essentially of a wash bufferand a hydrophilic porous material. In some embodiments, at least one oillayer is stabilized by a hydrophobic mesh (e.g. a hydrophobic syntheticmesh). For example, a first oil layer, second oil layer, a third oillayer, etc. may contain or consist essentially of a hydrophobicsynthetic mesh associated with the oil layer.

In some embodiments, the aqueous and oil phases or layers arestabilized, at least in part, by modulating one or more chemical orphysical material characteristics. For example, the aqueous and oilphases or layers may be stabilized by modulating geometry or one or morechemical or physical material characteristics including density, surfacechemistry, surface free energy, fluid retention, and/or porosity of ahydrophilic or hydrophobic porous material, if present in the system. Insome embodiments, the aqueous and oil phases or layers are stabilized inclose proximity to each other by suitable conditions such that fluidretention forces associating the fluid layer with the support structuredominate other forces (e.g. buoyancy) that might otherwise disrupt thefunctional layering or order of the phases. For example, in a simpletwo-phase system where water is introduced into a vessel over the top ofan oil that is less dense than water in the presence of gravity,buoyancy forces reorganize the system such that the oil will form alayer over the top of the water phase, relative to gravity. If ahydrophobic stabilizing porous material is present at the top surface ofthe oil, and is attached to the walls of the container prior tointroducing the water, fluid retention forces in porous materials canprevent buoyancy forces and forces/pressures arising from the act ofpouring from reordering the oil layer to the top position, keeping theoil below the aqueous layer.

In some embodiments, oil phase stabilization is adjusted, modified orselected by using oils of different densities. Oil phases may also beadjusted or modified by creating phases or layers where surface tensionand/or capillary forces dominate over forces arising from density and/oracceleration (e.g., gravity).

The density and/or surface properties of an aqueous phase or layer orpotentially the associated supporting structure can be adjusted toadjust the stability or association of a layer with the device or asupporting structure. In some embodiments, the densities and/or surfaceproperties of one or more aqueous phases or layers are adjusted ormodified by using aqueous phases or layers comprising different salt oramounts of salts, surfactants, etc. Surface properties of structuresassociated with the fluid can be adjusted as well, such as via oxygenplasma treatment of a polystyrene mesh to increase preference for fluidslike water. Aqueous phases or layers may also be modified by creatingphases or layers comprising heavy liquids. Heavy liquids include sodiumpolytungstate, sodium metatungstate, and the lithium metatungstate.These are all inorganic compounds, based on the [H2W12O40]6-polyanion,which is dissolved in water to form very dense solutions, which can bediluted for forming less dense aqueous phases or layers, but more thanpure water-based phases or layers. In some embodiments, aqueous phasesor layers are modified by creating phases or layers where surfacetension and/or capillary forces dominate over forces arising fromdensity and/or acceleration (e.g., gravity).

The density, mechanical properties, and/or surface properties of a phaseor layer in a system, device and method of the invention can also beadjusted with phase change, e.g. melting, boiling, sublimation, etc. ofmaterials. Useful phase change materials for adjusting or modifying thebuoyancy and/or surface tension in a phase or layer include polymericcompounds such as polyethylene glycols and methoxypolyethylene glycols.In some embodiments the phase change material can be a paraffin wax withan operational temperature above the melting temperature of the wax, forexample.

The porosity of a supporting structure can be adjusted or selected byusing specific materials of varying pore size, or differing pore sizeranges. Some useful porous materials (e.g., nitrocellulose) are madewith different pore sizes. Pore size may also be adjusted or modified insitu (e.g., using hydrogels that swell or degrade, or porous materialsladen with dried sugars).

In some embodiments, targets are positioned positively or negatively.Targets may be positioned positively, for example, by positioning orisolating a target (e.g., for detection or measurement) or removing atarget from a sample or material. Targets may be positioned negatively,for example, by positioning or removing one or more or all non-targets.In some embodiments, positioning a target by, for example, isolating,separating, moving or binding the target or a material bound to thetarget using a method, device or system of the invention is donepositively by positioning the target or a material connected to thetarget away from other materials, e.g., materials in a biological orother sample.

In some embodiments, positioning a target by, for example, isolating,separating, moving or binding the target or a material bound to thetarget using a method, device or system of the invention is donenegatively by removing non-target materials away from the target ofmaterial bound to the target.

In some embodiments of the invention using antibody-based cellisolation, for example, either positive or negative selection may beused. Cells that may be isolated using a method, device or system of theinvention, include, for example, stem cells, circulating fetal cells,circulating tumor cells, etc. The invention may also be used to isolaterare cells that may be masked within larger, more diverse backgrounds ofcells (e.g., the bloodstream, biopsy tissue, etc.) or bypatient-to-patient variation. Amongst other things, the methods, devicesand systems of the invention provide the means to separate rare targetcells from background, either positively or negatively.

Positive selection may utilize antibodies to capture cells in anantigen-dependent manner, yielding a captured population specific to achosen cellular marker (through antibodies, carbohydrate receptors,etc.). While precise, positive selection requires the marker to bespecific to the target population and known a priori. Negative selectionmay be used if distinguishing markers are unknown or non-differential(i.e., shared by neighboring cell populations), even if expressed atdiffering levels. Negative selection embodiments of the inventionleverage known non-target markers to deplete background populations. Inthis approach, the target cells remain uncaptured, enabling a negativeapproach to isolation. In the case of negative selection, the target ispositioned away from other materials by moving the other material awayfrom the target rather than moving the target itself (or the material towhich the target is bound).

The systems described herein comprise a container housing the variouscomponents of the system (e.g. the at least one aqueous phase (e.g.aqueous layer), the at least one oil phase (e.g. oil layer), theplurality of porous materials, etc.). Any suitable container may beused. The appropriate container may be selected based upon the desiredapplication of the system. Examples include, but are not limited to,test tubes, microcentrifuge tubes, dishes, slides, plates, multi-wellplates (e.g., 4-well, 8-well, 12-well, 96-well, 384-well, etc.), flasks,vials, channels, and the like. In some embodiments, the container is amulti-well plate, such that a plurality of samples may be processedsimultaneously.

The container may be any suitable size. In some embodiments, a smallcontainer (e.g. a multi-well plate) may be well-suited for isolation ofanalytes from biological samples. In other embodiments, a largercontainer may be well suited for isolation of analytes fromenvironmental samples, such as sewage samples. Any suitable volume ofaqueous and oil phases may be used, depending on the container selected.For example, for multi-well plates relatively small volumes of aqueousand oil phases may be used (e.g. less than 0.5 ml). However, as the sizeof the container increases, it is understood that the volume of aqueousand oil phases will scale appropriately.

In some embodiments, the system further comprises reagents for detectingthe target. In some embodiments, reagents for detecting the target arehoused toward, at or on a bottom surface of the container such that thesample passes through the plurality of porous materials prior tocontacting the reagents for detecting the target. For example, thereagents for detection of the target may be stabilized on a bottomsurface of the container by a suitable porous material. For example,reagents for detection of the target may be associated with ahydrophilic porous material (e.g., glass mesh, nylon) and positioned orstabilized below an oil phase or layer. Reagents may be stabilized aboveor on the bottom surface of the container by a structural porousmaterial, e.g. a hydrophilic porous material (e.g. glass mesh, nylon) insome embodiments. In some embodiments, reagents for detection of thetarget may not be associated with a supporting structure. For example,the reagents can be positioned below a stabilized oil phase or layer tohold them in place. In some embodiments, reagents are associated with asupporting structure (e.g., porous glass mesh, or non-porous materialdevice such as an nylon O-Ring) and positioned below a stabilized oilphase or layer. For example, reagents for detection of a target ortargets are retained on the bottom of the container by adding a suitablematerial on top of the reagents to hold them in place. In someembodiments, reagents for detection of a target or targets may be heldtoward, at or on the bottom surface of the container by placing afibrous material on top of the reagents (e.g. polypropylene mesh). Insome embodiments, the reagents are held on the bottom surface of thecontainer by a non-mesh or non-porous material device, for example, anO-Ring (e.g. PTFE O-Ring).

In other embodiments, reagents for detecting the target may be presentin a separate container from the container housing the at least oneaqueous phase and at least one oil phase. For example, the containerhousing the at least one aqueous phase, at least one oil phase, and thestacked porous materials may be placed on top or within a separatecontainer holding reagents for detecting the target. For example, thecontainer housing the at least one aqueous phase, at least one oilphase, and the porous materials may be used as an insert and placedwithin a separate container holding the reagents for detection of thetarget. The magnet may be placed below the container holding thereagents for detection of the target, such that the target-PMP complexesare drawn through the materials held within the insert and brought intocontact with the reagents for detecting the target. It will be notedthat in some described embodiments, the system contains at least oneaqueous phase or layer and at least one gaseous phase or layer, but nooil phase or layer, and that in other embodiments, the system containsat least one oil phase or layer and at least one gaseous phase or layer,but no aqueous phase or layer.

In some embodiments of the system, the magnet is a part of thecontainer. In some embodiments, including embodiments for disposableuses, for example, the magnet may be included or fixed within thecontainer (e.g. at the side of the container, at the bottom of thecontainer, etc.). In other embodiments of the system, the magnet may beattached or fixed to the outside bottom or side of the container. Inother embodiments of the system, the bottom of the container, or aportion of the bottom of the container, comprises a magnet. In otherembodiments of the system, the side of the container, or a portion ofthe side of the container, comprises a magnet.

In other embodiments, the magnet used is part of a fixture, instrument,holder, tool or the like that is used to position the magnet relative tothe PMI's.

In some embodiments, reagents for detection of the target comprisereagents for nucleic acid amplification (e.g. PCR, isothermalamplification, and the like) and/or sequencing. In some embodiments, thereagents for detecting the target comprise reagents for RT-PCR, qPCR,qtPCR, multiplex PCR, assembly PCR or asymmetric PCR, for example. Inother embodiments, the reagents for detecting the target comprisereagents for immunoassays, which may use antibodies and/or antibodyfragments to detect or measure a target or target analyte. In someembodiments, the immunoassay is an enzyme immunoassay, an ELISA(enzyme-linked immunosorbent assay, including direct ELISAs, indirectELISAs, sandwich ELISAs and competitive ELISAs), an IEMA(immunoenzymometric assay), a radioimmunoassay (RIA), afluoroimmunoassay, a chemiluminescent immunoassay (CLIA) and countingimmunoassay (CIA).

In some embodiments, the invention provides a disposable cartridgecomprising a flow-through assay to determine the presence or amount of atarget in a sample of a fluid comprising a sample application space, acartridge top, a cartridge bottom, reagents for detection orquantification of the target and an enclosure, the improvementcomprising employing within the enclosure target-binding paramagneticparticles, at least one aqueous phase and at least one oil phasestabilized in proximity to one another by inclusion of a porousstructural material associated with the aqueous phase or the oil phaseor both, and a magnet. Other phases, and/or alternative phases, may beused or included.

In some embodiments, the invention provides a flow assay device (e.g.,lateral flow, vertical flow) comprising a sample application portion, aconjugate portion, a test portion and pre-immobilized reagents indifferent parts of the device, the improvement comprising employingtarget-binding paramagnetic particles, at least one aqueous phase and atleast one gaseous or oil phase stabilized in proximity to one another byinclusion of a porous structural material associated with the aqueousphase or the gaseous or oil phase or both, and a magnet. Other phases,and/or alternative phases, may be used or included. In some embodiments,the improved flow assay device is designed for use as a disposable,point-of-care device or cartridge.

In some embodiments, the invention provides an immunometric assay todetermine the presence or concentration of a target substance in asample comprising forming a ternary complex of a first labeled bindingagent, said target substance, and a second binding agent said secondbinding agent being bound to a solid carrier wherein the presence oramount of the substance in the sample is determined by measuring eitherthe amount of labeled binding agent bound to the solid carrier or theamount of unreacted labeled binding agent, the improvement comprisingemploying target-binding paramagnetic particles, at least one aqueousphase and at least one oil phase stabilized in proximity to one anotherby inclusion of a porous structural material associated with the aqueousphase or the oil phase or both, and a magnet. Other phases, and/oralternative phases, may be used or included. In some embodiments, thesolid phase is a paramagnetic particle. in some embodiments one or moreof the binding agents is an antibody, an antibody fragment, anoligonucleotide, an aptamer, a peptide, a peptidomimetic, natural orchemically modified antisense oligonucleotides, or other suitable agentto assist with capture of a target. In other embodiments, the assayimproved with use of target-binding paramagnetic particles, at least oneaqueous phase and at least one oil phase stabilized in proximity to oneanother by inclusion of a porous structural material associated with theaqueous phase or the oil phase or is an IEMA, an RIA, a CIA, a CLIA or afluoroimmunoassay.

In some embodiments, reagents for detecting a target comprise reagentsfor identifying one aspect of the target. In some embodiments thereagents for detecting a target comprise reagents for identifying morethan one aspect of the target. Target aspects include, for example, apeptide, a protein, a glycoprotein, epigenetic modifications of anucleic acid, a nucleic acid sequence, cell surface receptor, a celltype, etc. In some embodiments the reagents for target detectioncomprise reagents for identifying more than one target, or one or moreaspects of one or more targets. In some embodiments, the reagents fordetecting more than one target are contained in different, physicallyseparated, portions of the system or device, or in different part of acontainer comprising a system or device of the invention. In someembodiments, multiple targets are isolated, and multiple types ofreagents for detecting these targets are contained within a singledevice or system. See, e.g., Example 13.

In some embodiments of the invention useful for performing one or moresteps of an assay for the detection or measurement of a target or targetanalyte, one or more of the phases or layers of the device or system maycomprise one or more of several different buffers. In some embodiments,one or more phases or layers comprise a coating buffer, a blockingbuffer, a stabilization buffer, a washing buffer, or act as or comprisea sample diluent. In some embodiments, antibodies or antibody fragmentsare used to generate a detection signal. In some embodiments, the assaycarried out using a device, system or method of the invention comprisesin magneto-actuated immunoassay in which the movement or positioning ofa target or target analyte is achieved using magnetic separation using amagnetic particle. In some embodiments, the particle used in theseembodiments is made of a core of magnetite that is chemically modifiedby the attachment of antibodies or antibody fragments. In someembodiments, one or more or all components of an assay are used toisolate or purify a target or target analyte.

In some embodiments, the reagents for detection of the target comprisereagents for loop-mediated isothermal amplification (LAMP)-baseddetection of the target. In general, LAMP reactions include a DNApolymerase with strong strand displacement activity and tolerance forelevated temperatures and up to six DNA oligonucleotides of a certainarchitecture. RT-LAMP reactions additionally include a reversetranscriptase. Samples with potential template molecules are added tothe reaction and incubated for 20 to 60 min at a constant temperature(e.g. 65° C.). The oligonucleotides act as primers for the reversetranscriptase, and additional oligonucleotides for the DNA polymeraseare designed so the DNA products loop back at their ends. These, inturn, serve as self-priming templates for the DNA polymerase. In thepresence of a few RNA template molecules, a chain reaction is set inmotion, which then runs until the added reagents (in particular, thedeoxynucleotide triphosphates) are used up.

LAMP assays or RT-LAMP assays are a particularly useful embodiment dueto their rapid nature, one-tube processing, and easy visualization ofresults without the need for expensive equipment or additionalmaterials. In particular embodiments, the reagents for detection of thetarget comprise reagents for a colorimetric assay for detecting thepresence amount the target. Such embodiments allow for a facilevisualization of whether or not the sample contains the target ofinterest. In some embodiments, the sample collection device containsreagents for a colorimetric loop mediated isothermal amplification(LAMP) assay. In embodiments wherein the nucleic acid is RNA, the samplecollection device may contain reagents for a colorimetric RT-LAMP assay.In some embodiments, the reagents for a colorimetric LAMP assay (orcolorimetric RT-LAMP assay) further include an indicator, which permitsevaluation of a color change in the sample in the presence of sufficientnucleic acid (e.g. the target nucleic acid which the LAMP or RT-LAMPreagents are designed to detect). Suitable indicators includepH-sensitive indicators and metal-sensitive indicators. In someembodiments, pH-sensitive indicators (e.g. phenol red) may be used, dueto their easy visualization with the naked eye. The best signaldetection approach for particular applications (e.g., enzyme(fluorometric, calorimetric, chemiluminescent, enhancedchemiluminescent), radiometric, direct fluorescent, time-resolvedfluorescent, direct chemiluminescent, phosphorescent, etc.) will bedetermined by the user. Signal amplification techniques and strategiesmay also be used in systems, devices, compositions and methods of theinvention, as may multiplex techniques.

In some embodiments, the reagents for detection of the target comprisereagents for a fluorescent assay for detecting the target, or fordetermining the amount of the target, either quantitatively,semi-quantitatively, or at a predetermined threshold amount. Forexample, the sample collection device may contain reagents for afluorescent LAMP or fluorescent RT-LAMP assay. Any suitable fluorescentdye may be used in a fluorescent LAMP or fluorescent RT-LAMP assay topermit a fluorescent signal to be generated in the presence ofsufficient nucleic acid.

In some embodiments, the reagents for detection of the target comprisereagents for a “Yes/No” assay.

In some embodiments, the reagents comprise oligonucleotides (e.g.primers) designed for detection of bacterial nucleic acid or nucleicacid from any life form or replicating unit, including nucleic acid fromeukaryotic cells, mitochondria and chloroplasts, etc. In someembodiments, the nucleic acid is bacterial nucleic acid. In someembodiments, the nucleic acid is viral nucleic acid. In someembodiments, the nucleic acid is nucleic acid from any source, includingsynthetic or genetically engineered source.

In some embodiments, the reagents comprise oligonucleotides designed fordetection of viral RNA. In some embodiments, the reagents compriseoligonucleotides designed for detection of, for example, a SARS-CoV2, acoronavirus, a rhinovirus, an influenza virus, a respiratory syncytialvirus, an adenovirus, a parainfluenza virus, a human immunodeficiencyvirus, a human papillomavirus, a rotavirus, a hepatitis virus (includinga hepatitis A, B, C, D and/or E virus), a zika virus, an Ebola virus, atuberculosis bacterium, Borrelia burgdorferi, Borrelia mayonii, astaphylococcus bacterium, an aspergillus fungus (including Aspergillusniger), or a streptococcus (including Streptococcus pyogenes). Forexample, the reagents may comprise oligonucleotides designed fordetection of a viral upper respiratory infection selected from aSARS-CoV2, a SARS, a coronavirus, a rhinovirus, an influenza virus, arespiratory syncytial virus, etc. In some embodiments, the reagentscomprise oligonucleotides for detection of a SARS-CoV-2 RNA or afragment thereof.

In some embodiments, the system further comprises a magnet. The magnetis used to draw the target-PMP complexes through the stacked porousmaterials and into contact with the reagents for detection of theanalyte, and therefore may be referred to herein as a “purificationmagnet”. The purification magnet may provide partial or completepurification. The purification magnet may be of suitable strength and/orplaced in a suitable proximity to the bottom of the container, or withinthe container, to draw some, most, substantially all, or all of thetarget-PMP complexes through the stacked porous materials. For example,the purification magnet may be positioned below the container. In someembodiments, the system comprises a plurality of purification magnets(e.g. arranged in an array). For example, a plurality of purificationmagnets may be used to address a plurality of containers (e.g. amulti-well plate containing multiple samples from which isolation anddetection of the analyte is desired) at the same time or in sequence. Insome embodiments, a second set of magnets are used to influence oradjust the uniformity and strength of the purification magnets. Forexample, when the purification magnets are arranged in an array pattern,a second set of magnets may be positioned around the perimeter of thearray to reduce edge effects, maintaining a more consistent magneticfield for each purification magnet in the array. Accordingly, a secondset of magnets may be referred to herein as “field stabilizationmagnets”. After binding, a magnet may be applied to the system, such asto the base of the container housing the plurality of porous materials,thus drawing target-PMP complexes through the stacked porous materialsto purify or substantially isolate the target from other componentswithin the sample. The pore size of the porous materials should besufficient to permit target-PMP complexes to pass through the pores,while preventing other undesired contaminants from passing. Undesiredcontaminants may be from any source, e.g., undesired components of anoriginal sample, environment, assay reagents, device, etc.

The pore size of the porous material may be optimized depending on thetarget to be isolated. In some embodiments, the pore size may range from0.5 μM-0.5 mm.

The systems described herein may be used to isolate a target from anydesired sample. In some embodiments, the sample is a biological sample.In some embodiments, the biological sample is a nasopharyngeal sample,an oropharyngeal sample, an oral swab or sponge sample, a nasal swabsample, a mid-turbinate sample, or a saliva sample. In particularembodiments, the biological sample is a saliva sample. In otherembodiments, the biological sample is an NP sample. In some embodiments,the sample is an environmental sample. For example, the sample may be asewage sample. In some embodiments, the environmental sample orbiological samples are crude samples and/or wherein the one or moretarget molecules are not purified or amplified from the sample prior toapplication of a method of the invention or manipulation with a device,system, method or composition of the invention.

In some embodiments, biological sample is first mixed with lysis/bindingbuffer with or with a solid phase or a lysis/binding buffer and a solidphase (e.g. PMPs), and then added to the system or device, which may bein a container. In some embodiments the system or device alreadycontains lysis/binding buffer and PMPs, and the biological sample isadded to it. In some embodiments involving a biological sample acquiredvia a swab (e.g. nasopharyngeal sample, an oropharyngeal sample, an oralswab sample, an oral sponge sample, a nasal swab sample, a mid-turbinatesample, etc.), the swab is submerged and mixed into the lysis/bindingbuffer (and PMPs if already contained in the container). In someembodiments, a biological sample is acquired using a separate device orcontainer which then interfaces with a container which already containslysis/binding buffer and solid phase, e.g., PMPs. The joining/mating ofthe two containers/devices facilitates biological sample introductioninto the system or device. In some embodiments, the biological sampleundergoes certain preprocessing or pretreatment steps before being addedto the system or device.

The sample may be collected and/or stored in a suitable container (e.g.a sample collection container) prior to adding the samples to a systemas described herein. Any type of sample collection container may be usedthat is suitable for receiving a sample and storing the sample. Examplesof sample collection containers include, but are not limited to, tubescontaining a reversibly removal cap, bags, syringes, droppers, and thelike. In some embodiments, the samples are pre-treated prior to use in asystem as described herein. For example, the samples may be pre-treatedin the sample collection container. As another example, the samples maybe moved to a suitable second container and pre-treated within saidsecond container.

In some embodiments, the samples may be pre-treated to inactivatepotential pathogens (e.g. virus, bacteria) within the sample. Forexample, the samples may be pre-treated prior to use in a system asdescribed herein. In some embodiments, the samples may be pre-treated tolyse cells within the sample, thus releasing the target (e.g. nucleicacid) for subsequent detection. In such embodiments, a pre-treatmentstep accomplishes both cell lysis (e.g. release of nucleic acid) andinactivation of potential pathogens within the sample. In someembodiments, the samples may be pre-treated by freezing, heating and/orthe addition of a denaturant to the sample. For example, the sample maybe pre-treated by heating to a sufficient temperature for a suitableduration of time to inactivate potential pathogens within the sample.For example, the sample may be heated to about 40° C. or higher. Forexample, the biological sample may be heated to about 40° C., 45° C.,50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., 85° C., 90° C.,95° C., 100° C., or more than 100° C. The sample may be maintained atthe heated temperature for a suitable duration of time, such as 5minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60minutes, or more than 1 hour. In particular embodiments, the sample maybe heated to 98° C.-100° C. for 5 minutes to accomplish both cell lysisand viral inactivation in a single heat treatment step. In someembodiments, pre-treating the sample comprises adding a denaturant toinactivate potential pathogens within the sample. For example, adenaturant may be present in the lysis buffer with which the sample iscontacted. For example, suitable denaturants include guanidine-baseddenaturants (e.g. guanidine hydrochloride, guanidine thiocyanate, etc.)and surfactants (e.g., Triton X-100, tween20). In some embodiments, thesample does not contain a denaturant. For example, in some embodimentsthe sample (e.g. saliva sample) may not contain a guanidine-baseddenaturant. In some embodiments, the sample (e.g. saliva sample)contains less than 0.3M of a guanidine-based denaturant. For example,the sample (e.g. saliva sample) may contain less than 0.3M, less than0.25M, less than 0.2M, less than 0.15M, less than 0.1M, or less than0.5M of a guanidine-based denaturant.

The viscosity of certain samples (e.g. saliva) makes sample handlingdifficult. Moreover, the viscosity of samples collected from differentindividuals varies, introducing potential issues with variability ofsample collection between subjects. For example, a saliva sample withhigh viscosity may result in less volume of saliva successfully beingpipetted into a desired container (e.g. for subsequent detection of apathogen in the sample) compared to saliva with decreased viscosity.This can introduce potential downstream issues for variations orinaccurate results, including false negative results. In someembodiments, the samples may be pre-treated to reduce viscosity of thesample and thereby improve sample handling in subsequent processingsteps. In particular embodiments, the pre-treatment step may beperformed to inactivate pathogen(s) within the sample and reduce theviscosity of the sample in one step. In some embodiments, one or moreagents to decrease viscosity may be added to the sample prior to usingthe sample in a system as described herein. In some embodiments, theagent to decrease viscosity is a reducing agent. Suitable reducingagents include, for example, dithiothreitol (DTT),tris(2-carboxyethyl)phosphine (TCEP), or 2-mercaptoethanol.

Any suitable amount of a reducing agent may be added to the sample (orpresent in the storage buffer in which the sample is placed uponcollection). In some embodiments, the reducing agent is present in thelysis buffer with which the sample is contacted. In some embodiments,suitable concentrations of reducing agents may range from 0-500 mM. Forexample, suitable concentrations of DTT or TCEP may range from 0-250 mM(e.g. OmM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM,about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM,about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM,about 210 mM, about 220 mM, about 230 mM, about 240 mM, or about 250mM). For example, dithiothreitol (DTT)) may be added to a biologicalsample (e.g. a saliva sample) at a suitable concentration to decreaseviscosity of the sample. In some embodiments, DTT may be added toachieve a 1× concentration within the saliva sample. As another example,suitable concentrations of 2-mercaptoethanol may range from 0-500 mM(e.g. 0 mM, about 25 mM, about 50 mM, about 75 mM, about 100 mM, about125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about250 mM, about 275 mM, 300 mM, about 325 mM, about 350 mM, about 375 mM,about 400 mM, about 425 mM, about 450 mM, about 475 mM, or about 500 mM.

In some embodiments, the viscosity reducing agent (e.g. DTT) is added tothe sample prior to heating the sample (e.g. to inactivate pathogensand/or induce cell lysis). In some embodiments, the viscosity reducingagent may be present in a sample storage buffer to which the sample isadded after collection. In some embodiments, the viscosity reducingagent is added to the sample after heating the sample. In someembodiments, the viscosity reducing agent is present in the lysis bufferwith which the sample is contacted. In some embodiments, freezing thesample may be performed to reduce the viscosity of the sample. Anysuitable pre-treatment step or combination of pre-treatment steps may beperformed to achieve the desired result (e.g. cell lysis, pathogeninactivation, and/or reduction of viscosity of the sample).

The sample may additionally comprise a suitable detergent. For example,the sample may comprise an ionic detergent (e.g. sodium dodecyl sulfate,deoxycholate, cholate, etc.), a non-ionic detergent (e.g. Triton X-100,DDM, digitonin, Tween 20, Tween 40, Pluronic F-127), a zwitterionicdetergent, or a chaotropic detergent. In some embodiments, the samplecomprises 0-5% detergent (v/v). For example, the sample may comprise 0%,about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%,about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%detergent. The detergent may be added to the sample (e.g. contacted withthe sample as part of a lysis buffer) and/or present in a sample storagebuffer to which the sample is added upon collection.

In some embodiments, the sample comprises a non-ionic detergent (e.g.Triton X-100). For example, the sample may comprise 0.001-.1% TritonX-100. The sample may be brought to a suitable volume for subsequent useby the addition of a suitable buffer. For example, the sample may bebrought to a suitable volume by the addition of phosphate bufferedsaline (PBS), universal transport medium (UTM), saline, and the like.Such buffers may be added to the sample or present in a sample storagebuffer to which the sample is added upon collection. The sample maycomprise one or more enzymes or chemical agents to assist with breakingdown the contents therein to facilitate release of the desired target.For example, the sample may comprise one or more enzymes, such as one ormore proteases. In particular embodiments, the sample may compriseproteinase K. The sample may additionally comprise one or more suitablereagents to prevent degradation of the target within the sample. Forexample, suitable buffers and/or inhibitors (e.g. RNase inhibitors,nuclease inhibitors, etc.) may be added to the sample prior to use in asystem as described herein.

The systems, devices, compositions and methods described herein may beused for isolation, detection, identification, or quantification of anydesired target from any sample or source. The devices, compositions andmethods of the invention may be used for positioning any desired targetfrom any sample or source for any purpose, including detection,quantification, etc.

In some embodiments, the devices, systems and/or methods are used forisolation and subsequent detection of a desired target. In someembodiments, the target is a cell. In some embodiments, the target is anucleic acid (e.g. DNA, RNA, or various subtypes thereof including mRNAand rRNA), a protein, a metabolite, a carbohydrate, a glycopeptide, or alipid. For example, the target may be DNA or RNA. In some embodiments,the target may be nucleic acid or proteins (e.g. antibodies, hormones,etc.) resulting from a pathogen infecting one or more subjects fromwhich the sample was obtained. For example, the target may be bacterialnucleic acid (e.g. bacterial DNA or RNA) or viral nucleic acid (e.g.viral DNA or RNA). As another example, the target may be antibodiesproduced by the subject in response to infection with a pathogen.

In some embodiments, the devices, systems and/or methods are used todetermine identity or paternity by sample analysis. In some embodiments,a sample is provided for use in device, system and/or method of theinvention for prenatal or postnatal screening.

In some embodiments, the sample is obtained from a subject suspected ofhaving an infection. For example, the biological sample may be obtainedfrom a subject suspected of having an infection. In some embodiments, anenvironmental sample may be obtained from an area in which one or moremembers of the population are suspected of having an infection. Forexample, sewage may be collected and used to determine whether one ormore members in the surrounding population have an infection ofinterest. The subject or one or more members of the population may besuspected of having any infection by a pathogen that can be detected inthe sample, or an infection which causes the person to produceantibodies which may be detected in the sample. In some embodiments, thesubject from which a biological sample is obtained or one or morepersons in a population proximal to an area from which an environmentalsample is collected are suspected of having a SARS-CoV2, a coronavirus,a rhinovirus, an influenza virus, s respiratory syncytial virus, anadenovirus, a parainfluenza virus, a human immunodeficiency virus, ahuman papillomavirus, a rotavirus, hepatitis virus (including ahepatitis A, B, C, D and/or E virus), a zika virus, an Ebola virus, atuberculosis bacterium, Borrelia burgdorferi, a staphylococcusbacterium, an aspergillus fungus (including Aspergillus niger), or astreptococcus (including Streptococcus pyogenes). In some embodiments,the subject or a member of the population may be suspected of having abacterial infection or a viral infection. For example, the subject or amember of the population may be suspected of having an upper respiratoryinfection. For example, the subject or a member of the population may besuspected of having a viral upper respiratory infection, includinginfection with SARS-CoV-2, a coronavirus, rhinovirus, influenza,respiratory syncytial virus, and the like.

The systems described herein find use in methods for positioning orisolating a target from a sample, and/or the detection, identification,quantification or purification (partial or complete) of a target. Insome embodiments, the systems described herein find use in methods forisolating and subsequently detecting the target in the sample. Forexample, in some aspects provided herein is a method for isolating atarget from a sample, comprising adding the sample to a system asdescribed herein. In some embodiments, the sample is lysed by cominginto contact with a lysis buffer, thereby releasing the target analyte.The method further comprises applying a magnetic force to the bottom orside of a container, depending on the orientation of the phases orlayers, meshes, etc., thus drawing the target analyte through theplurality of porous materials, thereby purifying the target from otherpotential contaminants present in the sample. In some aspects, providedherein is a method for isolating and detecting a target in a sample. Themethod comprises adding the sample to a system comprising a containerhousing a plurality of porous materials and reagents for detection ofthe target as described herein. The method further comprises applying amagnetic force to the bottom or side of the container, thereby drawingthe target through the plurality of porous materials and into contactwith reagents for detection of the target housed on a bottom surface ofthe container.

In some embodiments of the methods described herein, the sample iscontacted with paramagnetic particles or functionalized paramagneticparticles (PMPs) as described herein prior to applying the magneticforce to the system. Contacting the sample with the paramagneticparticles generates one or more target-PMP complexes, and applying themagnetic force to the system draws the target-PMP complexes through theplurality of porous materials towards a bottom surface of the container.In some embodiments, the sample is contacted with (e.g. mixed with)paramagnetic particles in a separate container to generate a compositioncomprising one or more target-PMP complexes, and the composition issubsequently placed (e.g. pipetted into) the container housing theplurality of porous materials. In other embodiments, the paramagneticparticles are housed in the container housing the plurality of porousmaterials and the aqueous and oil phase(s). For example, lyophilizedparamagnetic particles may be present within the container. Theparamagnetic particles may be present within the container in liquidform (e.g. as part of a lysis buffer). In such embodiments, adding thesample to the container will cause the sample to contact the PMPs,thereby generating the target-PMP complexes within the container itself.

In some embodiments, the sample is contacted with a lysis buffer asdescribed herein. As described above, the lysis buffer may be contactedwith the sample prior to adding the sample to the system or the lysisbuffer may be present within the container housing the plurality ofporous materials. Contacting the sample with the lysis buffer enablesrelease of the target from the various components of the sample, therebyfacilitating subsequent isolation and/or detection of the target.

In some embodiments, the sample is contacted with a wash buffer. Asdescribed above, the wash buffer may be present within the containerhousing the plurality of porous materials. Application of the magneticforce to the bottom surface of the container thereby draws the target(e.g. target-PMP complexes) through the wash buffer present within thecontainer, thereby facilitating further purification of the target.

In some embodiments, the methods further comprise detecting the targetfollowing removal or isolation of some, all or substantially all (asdesired or required for purposes of the method), from the sample. Insuch methods, the systems comprise reagents for detecting the targethoused at, near or on a bottom (or side) surface of the container, asdescribed herein. The target-PMP complexes are drawn through the aqueousand oil phases, and through the plurality of porous materials, and comeinto contact with the reagents for detection of the target. In someembodiments, a suitable incubation time is allowed to pass at a suitabletemperature (e.g. 20-60 minutes at 65° C.) and a signal resulting fromcontact is measured. For example, a colorimetric signal (e.g. a colorchange) or a fluorescent signal may be measured to determine which wellscontain the target. Measuring a signal (e.g. color change, fluorescentsignal) may occur, for example, by visualization (e.g. by the nakedeye). Alternatively, the signal may be measured using equipment, such asa plate reader. For example, a fluorescent signal may be measured usinga plate reader. In some embodiments, the isolated target-PMP complexesare contacted with the reagents for LAMP-based detection of the target,and a signal resulting from contact is measured. For example, the signalmay be a colorimetric signal (e.g. a signal from a colorimetric RT-LAMPassay) or a fluorescent signal (e.g. a signal from a fluorescent RT-LAMPassay).

In some embodiments, the methods described herein are performed on asingle sample. In other embodiments, the methods are performedsimultaneously on a plurality of samples. In some embodiments, samplesmay be pooled and subsequently used in the systems and methods describedherein. For example, biological samples may be collected from aplurality of distinct individuals, pooled together, and used in themethods described herein to determine whether a population has cases ofinfection with a pathogen (e.g. with SARS-CoV2). As another example, aplurality of biological samples may be collected from an individual, andthe plurality of biological samples from a distinct individual may bepooled to increase the amount of sample available to be used in themethods described herein. Such embodiments may be useful for instanceswhere an individual may be unable to provide an adequate volume ofsaliva during one collection, or when multiple tests may be performedusing the same sample.

In some embodiments, the method steps described herein are automated. Insome embodiments, sample preparation steps described herein areautomated. In some embodiments, detection steps described herein areautomated. In some embodiments, acquisition of results is automated. Insome embodiments, communication of results to other devices or non-userthird parties is automated. In some automated embodiments, the stepsand/or methods described herein are executed by a computer, wherein thecomputer comprises a processor and a memory. The memory may containsoftware which instructs the processor to execute a given task. Forexample, the memory may contain software which instructs the processorto cause a multichannel pipette to attach pipette tips to the pipette,aspirate a sample, mix the biological sample with a PMPs to generate acomposition comprising one or more target-PMP complexes, aspirate thecomposition into a system as described herein, bring a magnet intoproximity to a bottom or other surface of the container (e.g. a sidesurface) housing the sample, or turning on an electromagnet that iswithin or in proximity of a surface of the container, and a plurality ofporous materials, and other necessary functions to perform the claimedmethod.

The inventions described herein include a system for isolating a targetfrom a sample, the system comprising at least one aqueous phase and atleast one oil phase stabilized in proximity to one another within acontainer. In some embodiments of this system, the at least one aqueousphase and the at least one oil phase are stabilized within the containerby a hydrophilic porous material immersed within the at least oneaqueous phase and/or a hydrophobic porous material immersed within theat least one oil phase. In some embodiments of this system, the at leastone aqueous phase and the at least one oil phase are stabilized withinthe container by modulating one or more chemical or physical materialcharacteristics selected from buoyancy, surface chemistry, and porosityof a hydrophilic/hydrophobic porous material, if present in the system.In some embodiments, the at least one aqueous phase and the at least oneoil phase are stabilized within the container by a hydrophilic porousmaterial immersed within the at least one aqueous phase, a hydrophobicporous material immersed within the at least one oil phase, and bymodulating surface chemistry such that the buoyancy of the at least oneoil phase is less than the surface tension of the at least one aqueousphase. In any of these embodiments, the system may comprise a firstaqueous phase, a second aqueous phase, a first oil phase, and a secondoil phase. In any of these embodiments of a system described herein, thephases may be stacked in an alternating fashion within the container,such that the first and second aqueous phase are not in direct contactwith one another and the first and the second oil phases are not indirect contact with one another. In any of these embodiments, the systemis provided in a device comprising a container, and the container maycomprise a top opening to permit addition of a sample to the container.In any of these embodiments, the system is provided in a devicecomprising an insert, and the insert may comprise an opening to permitaddition of a sample to the insert. In some embodiments, the at leastone aqueous phase is closest to the top opening of the container. Insome embodiments, the at least one aqueous phase is closest to theportion of the insert where a sample is added. In some embodiments, theat least one oil phase is closest to the top opening of the container.In some embodiments, the at least one oil phase is closest to theportion of the insert where a sample is added. In any of theseembodiments, the at least one aqueous phase may comprise, consistessentially of, or consist of a lysis buffer. In any of theseembodiments, the at least one aqueous phase may comprise, consistessentially of, or consist of a wash buffer. In any of theseembodiments, the system, device, container or insert may compriseparamagnetic particles (PMPs). In some embodiments, the PMPs are housedwithin the container. The system of claim the PMPs are lyophilized ordried, or in a liquid form. In some embodiments, PMPs are housed withinthe at least one aqueous phase. In some embodiments with more than oneaqueous phase or layer, PMPs are housed within more than one or all ofthe aqueous phases or layers. In some embodiments, PMPs are housedwithin the at least one oil phase or layer. In some embodiments withmore than one oil phase or layer, PMPs are housed within more than oneor all of the oil phases or layers. In any of these embodiments, thesystem further comprises a magnet or other device to provide a magneticforce.

The inventions described herein include a system for isolating a targetanalyte from a sample, the system comprising a first aqueous phase, asecond aqueous phase, a first oil phase, and a second oil phase wherein(a) the phases are stacked in an alternating fashion within a container,such that the first and second aqueous phases are not in direct contactwith one another and the first and the second oil phases are not indirect contact with one another, and (b) the phases are stabilizedwithin the container by (i) a hydrophilic porous material immersedwithin the first aqueous phase, (ii) a hydrophilic porous materialimmersed within the second aqueous phase; (iii) a hydrophobic porousmaterial immersed within first oil phase; and (iv) a hydrophobic porousmaterial immersed within the second oil phase.

In some embodiments of this system, the phases are further stabilizedwithin the container by modulating surface chemistry such that thebuoyancy of each oil phase is less than the surface tension of eachaqueous phase, and/or such that the buoyancy of each oil phase is lessthan the water retention of each hydrophilic porous material. In someembodiments, the container comprises a top opening to permit addition ofa sample to the container. Either the first aqueous phase or layer orthe first oil phase or layer may be closest to the top opening of thecontainer. The first aqueous phase or layer may comprise or consistessentially of a lysis buffer. In some embodiments of this system, thesecond aqueous phase comprises a wash buffer. In some embodiments ofthis system, the system further comprises PMP. In some embodiments ofthis system, the PMPs are housed within the container. In someembodiments, the PMPs may be in lyophilized or dried or liquid form. Insome embodiments, the PMPs are housed within the first aqueous phase orthe second aqueous phase or both. In some embodiments, the PMPs arehoused within the first oil phase or the second aqueous phase or both.In any of these embodiments, the system further comprises a magnet orother device to provide a magnetic force. In some embodiments, thecontainer comprises a multi-well plate. In some embodiments, thecontainer comprises an insert. In some embodiments, the insert can beinserted into a multi-well plate. In some embodiments, the containercomprises a single-use device.

Systems, devices, methods and compositions of the invention may be usedfor (and include reagents for) moving, isolating (in whole or in part),purifying (in whole or in part), detecting and/or quantifying a target.Targets and/or analytes include, for example, small molecules, proteins,peptides, immunoglobulins (e.g. IgA, IgM, IgG, IgE, lambda light chain,kappa light chain), enzymes, lipids, receptors (e.g. Her2 receptor),nucleic acids (e.g. DNA, introns, exons, non-coding elements, RNA, rRNA,mRNA, microRNA), circulating tumor DNA (ctDNA), orphan non-coding RNA(oncRNA), circulating pathogen DNA and circulating pathogen RNA,antigens (e.g. PSA), hormones (e.g. testosterone) and cancer and othercells, including circulating tumor cells (e.g., circulating tumor cellsof epithelial origin which are related to metastatic breast, prostate,and colorectal cancers), circulating endothelial cells, cellularvesicles, exosomes, bacterial quorum sensing molecules. Targets andanalytes include biomarkers, including molecular and histologicbiomarkers, screening markers (primary, secondary and targeted),diagnostic biomarkers, prognostic biomarkers, predictive biomarkers,pharmacodynamic/response biomarkers, susceptibility/risk biomarkers,monitoring biomarkers and safety biomarkers.

Any bacterial, virus or other pathogen may be tested, isolated,separated, purified, identified, detected or quantified using a system,device, method or composition of the invention. In some embodiments, aviral target for testing, isolation, separation, purification, detectionor quantification is a Coronaviridae virus, a Picornaviridae virus, aCaliciviridae virus, a Flaviviridae virus, a Togaviridae virus, aBornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, aRhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, ora Deltavirus. In other embodiments, the virus is a Coronavirus, a SARS,a Poliovirus, a Rhinovirus, a Hepatitis A virus, a Norwalk virus, aYellow fever virus, a West Nile virus, a Hepatitis C virus, a Denguefever virus, a Zika virus, a Rubella virus, a Ross River virus, aSindbis virus, a Chikungunya virus, a Borna disease virus, an Ebolavirus, a Marburg virus, a Measles virus, a Mumps virus, a Nipah virus, aHendra virus, a Newcastle disease virus, a Human respiratory syncytialvirus, a Rabies virus, a Lassa virus, a Hantavirus, a Crimean-Congohemorrhagic fever virus, an Influenza virus, or a Hepatitis D virus. Insome embodiments, the virus is one or more of the above viruses (oranother virus) that has evolved or mutated to a new strain. In someembodiments, the virus is a virus that has been created, mutated orengineered by humans.

In some embodiments, the invention provides a method for monitoring orevaluating viral disease outbreaks and/or viral evolution using asystem, device, method or related composition of the invention.

In some embodiments, devices, systems, methods and compositions of theinvention are used in a method of screening samples for viral antigens,viral nucleic acids and/or viral specific antibodies, bacterial and/orother pathogen-specific antigens, nucleic acids and/or antibodies.

In some embodiments, a nuclease inactivation step is carried out in orwith a device, system, method or composition of the invention inassaying, testing for, screening for, separating, isolating, purifying,identifying, detecting and/or quantifying a target nucleic acid. Someembodiments include heat inactivation, chemical inactivation, ultrasonicinactivation, etc. In some embodiments, targets for tests or assays orother protocols using systems, devices, methods and compositions of theinvention include tumor markers (e.g., alpha-fetoprotein (AFP),beta-2-microglobulin (B2M), beta-human chorionic gonadotropin (β-hCG),bladder tumor antigen (BTA), chromogranin A (CcA, neuroendocrinetumors), gastrin (gastrinoma), 5-HIAA (carcinoid tumors) ALK generearrangements and overexpression, BCL2 gene rearrangements, BRCA1 andBRCA2 gene mutations); cancer genes and subsequences; cancer markers,including, for example, programmed death ligand 1; ER/PR, CA15-3 andCA27.29 (breast cancer); EGFR, KRAS and UGT1A1 (colorectal cancer);HER-2/neu (breast and gastric cancers); c-KIT/CD117 gastrointestinalstromal tumor, mucosal melanoma, acute myeloid leukemia, and mast celldisease); CD20, CD30, FIP1L1-PDGFRalh, Philadelphia chromosome,PML/RAR-alpha, TPMT, UGT1A1 (leukemia, lymphoma); EML4/ALK, EGFR, KRAS(lung cancer), BRAF (melanoma), CA125, CA125 II and HE4 (ovariancancer); BRAF V600 mutations (e.g., cutaneous melanoma, colorectalcancer, and non-small cell lung cancer); CA19-9 and CA19-9 XR(pancreatic, gallbladder, bile duct, and gastric cancers); calcitonin(medullary thyroid cancer); carcinoembryonic antigen (CEA) (colorectalcancer and other cancers; CD19 and CD22 (B-cell lymphomas andleukemias); CD20 (non-Hodgkin lymphoma); CD25 (non-Hodgkin (T-cell)lymphoma); CD30 (classic Hodgkin lymphoma, B-cell and T-cell lymphomas);CD33 (acute myeloid leukemia); chromosome 17p deletion (chroniclymphocytic leukemia); chromosomes 3, 7, 17, and 9p2 (bladder cancer);nuclear matrix protein 22, fibrin/fibrinogen (bladder cancer);cytokeratin fragment 21-1 (lung cancer); cyclin D1 (CCND1) generearrangement or expression (lymphoma, myeloma); Des-gamma-carboxyprothrombin (DCP)(hepatocellular carcinoma); gene mutations (e.g. DPD,EGFR, FGFR2, FGFR, FLT3, IDH1, IDH2, JAK2, KRAS and MYD88 genemutations); gene rearrangements (e.g., IRF4 gene, ROS1 gene, and T-cellreceptor gene rearrangements); gene fusions (e.g., NTRK gene fusion andPML/RARα fusion gene); PCA3 mRNA, PSA free and PSA total (prostatecancer); HER2/neu gene amplification or protein overexpression (breast,ovarian, bladder, pancreatic, and stomach cancers); lactatedehydrogenase (germ cell tumors, lymphoma, leukemia, melanoma, andneuroblastoma); MYC gene expression, myeloperoxidase (MPO), terminaltransferase (TdT)(lymphomas, leukemias); neuron-specific enolase(NSE)(neuroblastoma); and, e.g., prostatic acid phosphatase(PAP)(metastatic prostate cancer), tumor suppressors lost in cancer(e.g. BRCA1, BRCA2); cardiovascular and cardiometabolic markers (e.g.C-reactive protein (CRP), troponins, including high-sensitivity cardiactroponin I and cardiac troponin T (e.g., cTnI and cTnT), B-typenatriuretic peptides (e.g., BNP and NT-proBNP), D-dimer, tetranectin,serum cyclin-dependent kinase 9), CK-MB, galectin-3, adiponectin,adipocyte fatty acid-binding protein, heart-type fatty acid-bindingprotein, lipocalin-2, fibroblast growth factor 19 and 21,retinol-binding protein 4, plasminogen activator inhibitor-1,25-hydroxyvitamin D, and proprotein convertase subtilisin/kexin type 9(PSCK9), lipocalin-2, H-FABP, A-FABP), triglycerides, high-densitylipoprotein (HDL)-cholesterol, and low-density lipoprotein(LDL)-cholesterol, etc.; growth factors (e.g., TGFβ, FGF-19, FGF-21,EGF, PDGF); and, inflammatory biomarkers (e.g., interferons andcytokines (e.g. TNFα, IL-1, IL-6 and other interleukins), alpha-1antitrypsin, alpha-1 glycoprotein, anti-CCP, ASO (anti-streptolysin),complement C3, complement C4, CRP, IgA, IgE, IgG, IgM), procalcitonin,PCT (BRAHMS), rheumatoid factor), chemokines (e.g., G-CSF, GM-CSF),RPB-4, PAI-1, 25-hydroxyvitamin D, etc.). In some embodiments, targetsinclude hormones, amyloids and other receptors (e.g., IFN receptors,IL-6 receptors, IL-10 family receptors, TGFβ family receptors, chemokinereceptors); protein signatures (e.g., 5-Protein signature (OVA1)) andgene signatures (e.g., 17-, 21-, 46- and 70-gene signatures). In otherembodiments, targets include disease vectors, including bacteria,viruses and fungi. In some embodiments, targets include bacterial, viraland/or fungal nucleic acids, alone, together or in multiplex format.

Other targets include active B-12, B12, ferritin, folate, haptoglobin,homocysteine, iron, transferrin and UIBC (unsaturated iron-bindingcapacity). These targets may be used, for example, in assays or testsfor anemia.

Other targets include active alkaline phosphatase, calcium, intact PTH(intact PTH), magnesium, phosphorous and vitamin D. These targets may beused, for example, in assays or tests for bone disease or disorders,including evaluation of bone remodeling and the identification ofdisorders involving mineral pathways that impact bone formation.

Other targets include targets for use in tests or assays to test formany types of cancers including, breast, colon, gastrointestinal, liver,ovarian, pancreatic, testicular and prostate cancer, and others asnoted. Additional cancer-related targets include CYFRA 21-1 (cytokeratin19 fragment), pepsinogen I and pepsinogen II, and PIVKA-II (acirculating precursor of prothrombin and hepatocarcinoma marker), andproGRP (progastrin-releasing peptide) and SCC (squamous cell carcinomaassociated antigen.

Other targets include targets for use in tests or assays to test formetabolic diseases impacting glucose function, including diabetes. Theyinclude C-peptide, creatinine, creatinine (enzymatic), fructosamine,glucose, hemoglobin A1c, insulin and microalbumin.

Other targets include targets for use in tests or assays to test for thepresence of abused drugs and toxic levels of prescription medications.Targets include acetaminophen, amphetamine/methamphetamine,barbiturates, benzodiazepines, cannabinoids, cocaine, ecstasy,methadone, ethanol, methanol, opiates, PCP (phencyclidine), salicylate,and antidepressants, including tricyclic antidepressants.

Other targets include targets for use in reproductive endocrinologytests or assays to evaluate fertility and/or pregnancy status. Theyinclude DHEA-S, estradiol, FSH, hCG (including total beta-hCG), LH(luteinizing hormone), progesterone, prolactin, SHBG (sex hormonebinding globulin), testosterone (free testosterone, attachedtestosterone and/or total testosterone). Assays that may be carried outusing systems, devices, methods and compositions of the inventioninclude that testosterone 2^(nd) generation assay.

Other targets include targets for use in infectious disease tests orassays. They include CMV IgG, CMV IgM, CMV IgG avidity, rubella IgG,rubella IgM, toxoplasma IgG, toxoplasma IgM, toxoplasma IgG avidity.

Targets also include hepatitis targets, including anti-HAV IgG, anti-HAVIgM, anti-HBc IgM, anti-HBe, anti-HBs, anti-HCV, HBeAg (including HBsAgQuantitative and Qualitative) and HCVAg. Targets for other infectiousdiseases include chagas (caused by the parasite Trypanosoma cruzi), EBVEBNA-1-IgG, EBV VCA IgG, EBV VCA IgM, syphilis TOP. Other targetsinclude anti-HTLV-I/HTLV-II (retrovirus).

Other targets include targets for use in tests or assays using systems,devices, methods and compositions of the invention to evaluate ordiagnose hepatic function and/or liver disease. These targets includealbumin (BCB and BCP), alkaline phosphatase, alpha-1 antitrypsin, ALT(alanine aminotransferase), ALT, activated (alanine aminotransferase),ammonia, AST (aspartate aminotransferase), AST, activated (aspartateaminotransferase), bile acids, cholinesterase, cholinesterase/dibucain,direct bilirubin, total bilirubin, GGT (gamma-glutamyl transferase),lactate dehydrogenase and PIVKA-II (des-gamma-carboxy prothrombin).

Other targets include targets for use in tests or assays using systems,devices, methods and compositions of the invention to evaluate ordiagnose traumatic brain injury (mTBI [UCH-L1+GFAP]).

Other targets include targets for use in tests or assays using systems,devices, methods and compositions of the invention to evaluate ordiagnose thyroid disorders. They include anti-thyroid peroxidase(anti-TPO) and anti-thyroglobulin (anti-TG) antibodies, free T3(triiodothyronine), total T3, free T4 (thyroxine), total T4, TSH(thyroid stimulating hormone) and T-uptake (thyroid hormone uptake,which provides information on the number of thyroid hormone bindingsites, consisting primarily of thyroid binding globulin, thyroxinebinding prealbumin and albumin).

Other targets include targets for use in tests or assays using systems,devices, methods and compositions of the invention to evaluate ordiagnose renal diseases or disorders. These targets includebeta-2-microglobulin, creatine, creatine (enzymatic), cystatin C,microalbumin, NGAL (neutrophil gelatinase-associated lipocalin), protein(urine/CSF), urea nitrogen and uric acid.

Other targets include targets for use in tests or assays using systems,devices, methods and compositions of the invention to help preventrejection and reduce toxicity in transplant patient. Targets includecyclosporine, sirolimus and tacrolimus.

Other targets include the proteins apolipoprotein A1, apolipoprotein B,transferrin, ceruloplasmin, haptoglobin, Lp(a) and prealbumin.

Other targets include any therapeutic drug(s) for treatment monitoringand precision medicine, e.g., for helping make medical decisions,treatments, practices, or products tailored to a subgroup(s) ofpatients. Targets for monitoring or evaluation of use in, therapeuticactivity in, or suitability for a subject include any marketedtherapeutic or therapeutic candidate (including clinical trialcandidates). They include, for example, amikacin, digitoxin, digoxin,lithium, methotrexate, steroids (e.g. progesterone), phenytoin,quinidine, theophylline, anticonvulsants (e.g. valproic acid),antifungals, antivirals and antibiotics (e.g. tobramycin, vancomycin).

In some embodiments, a device, system, method or composition is used in(or as) a diagnostic method. Diagnostic methods include but are notlimited to diagnostic methods, assays and tests for targets, includingpathogens, cardiovascular and neurological events, and diseases,disorders and conditions including cancers (e.g. early detection ofcancer), and other methods, assays and tests directed to any of thetargets disclosed or referred to herein.

Devices, systems, methods and compositions of the invention can be usedin or with any assay format or device. Formats include direct, indirectand sandwich assays which are run manually or semi-automated onmulti-well plates (e.g., 8-, 24-, 48-, 96- and 384-well plates) wheresamples are measured in duplicate, for example. They include anyimmunoassay, including any of the immunoassays described or referred toherein. They include the use of devices, systems, methods andcompositions of the invention in or for any ligand-binding assay thatmeasures binding between a ligand and a receptor, any immunoassay thatdetects antibody-antigen binding, and any bioassay that measuresbiological activity in response to certain stimuli.

In some embodiments, the invention comprises any assay or assay deviceor assay format, the improvement comprising a fluid-fluid interfaceand/or fluid phase or layer stabilized with an associated supportingstructure (e.g. a porous mesh) having preference for at least one fluid.

In some embodiments, reagents for detection or quantification or atarget may be housed at, near or on a bottom surface of a container. Insome embodiments, the reagents for detecting the target comprisereagents for a loop mediated isothermal amplification (LAMP) or areverse transcriptase loop mediated isothermal amplification (RT-LAMP)assay. In some embodiments, the LAMP or RT-LAMP assay is a colorimetricassay or a fluorescent assay.

The inventions include the use of a system or device in a method forisolating a target from a sample. Another embodiment of the inventioncomprises a method for isolating a target from a sample, the methodcomprising (a) adding a sample to a system comprising at least oneaqueous phase and at least one oil phase stabilized in proximity to oneanother within a container; and (b) applying a magnetic force to thesystem, wherein the sample is contacted with paramagnetic particles(PMPs) prior to applying the magnetic force to the system, whereincontacting the sample with the paramagnetic particles generates one ormore target-PMP complexes, and wherein applying the magnetic force tothe system draws the target-PMP complexes through the at least oneaqueous phase and the at least one oil phase towards a bottom surface ofthe container. In some embodiments of the method, the at least oneaqueous phase and the at least one oil phase are stabilized within thecontainer by a hydrophilic porous material immersed within the at leastone aqueous phase and/or a hydrophobic porous material immersed withinthe at least one oil phase and/or by modulating one or more chemical orphysical material characteristics selected from buoyancy, surfacechemistry, and porosity of a hydrophilic/hydrophobic porous material, ifpresent in the system. In some embodiments of the method, the methodcomprises use of a system two or more aqueous phases or layers, and twoor more oil phases or layers, which may or may not be stacked in analternating fashion within the container, such that, for example, afirst and second aqueous phase are not in direct contact with oneanother and a first and second oil phase are not in direct contact withone another. Other embodiments relating to containers, inserts, PMPs,wash buffers, lysis buffers and so on, as well as samples and reagents,are as above.

In another method embodiment of the invention isolating a target from asample, the method comprises (a) adding a sample to a system comprisinga first aqueous phase, a second aqueous phase, a first oil phase, and asecond oil phase, wherein the phases or layers are stacked in analternating fashion within the container, such that the first and secondaqueous phases are not in direct contact with one another and the firstand the second oil phases are not in direct contact with one another,and the phases or layers are stabilized within the container by ahydrophilic porous material immersed within the first aqueous phase, ahydrophilic porous material immersed within the second aqueous phase, ahydrophobic porous material immersed within first oil phase; and ahydrophobic porous material immersed within the second oil phase; and(b) applying a magnetic force to the system, wherein the sample iscontacted with paramagnetic particles (PMPs) prior to applying themagnetic force to the system, wherein contacting the sample with theparamagnetic particles generates one or more target-PMP complexes, andwherein applying the magnetic force to the system draws the target-PMPcomplexes through the phases towards a bottom surface of the container.In some embodiments of this method, the phases are further stabilizedwithin the container by modulating surface chemistry such that thebuoyancy of each oil phase is less than the surface tension of eachaqueous phase, and/or such that the buoyancy of each oil phase is lessthan the water retention of each hydrophilic porous material. In someembodiments of this method, the container comprises a top opening topermit addition of a sample. In some embodiments of this method, thefirst aqueous phase or layer or the first oil phase or layer is closestto the top opening of the container. In some embodiments of this method,the first or second aqueous phase or layer, or both, comprises orconsists essentially of a lysis buffer. In some embodiments of thismethod, the first or second aqueous phase or layer, or both, comprisesor consists essentially of a wash buffer. In some embodiments of thismethod, the first or second aqueous phase or layer comprises or consistsessentially of a lysis buffer and the first or second aqueous phase orlayer comprises or consists essentially of a wash buffer. In someembodiments of this method, all PMPs are housed within the container. Insome embodiments of this method, all PMPs are housed within one or moreaqueous and/or oil phases or layers. In other embodiments of thismethod, some PMPs are housed within the container and some are added tothe sample or the container or both during the method. In someembodiments, the sample is biological sample, an environmental sample(e.g., a sewage sample), a saliva sample, a swab sample, a sampleobtained from a subject suspected of having an infection. In someembodiments, the subject is suspected of having a viral infection, aviral upper respiratory infection, or, for example, an infectionselected from SARS-CoV2, SARS, a coronavirus, rhinovirus, influenza, andrespiratory syncytial virus. In certain embodiments, the targetcomprises viral nucleic acid. In some embodiments, the target comprisesSARS-CoV-2, hepatitis B, hepatitis C, HIV, West Nile Virus, herpesand/or influenza nucleic acid.

In some embodiments, the fluid phases and layers, including aqueous, gasand oil phases and layers, stabilizing structures and other componentsdescribed herein are designed and incorporated together in a holdingbody (e.g., a vessel, container, insert, etc.) to form the systems,devices and methods using certain predefined design guidelines. Thedesign guidelines for each component can be dependent upon one or morefactors such as, e.g., holding body design (i.e., single-piece body,multiple piece body, modular body, single read chamber, multiple readchamber, and the like), manufacturing process (e.g., injection molding,blow molding, hot stamping, casting, machining, etc.), phases and layers(e.g., aqueous, oil, gas, blends, mixtures and emulsions, etc.),structural materials (e.g., polypropylene mesh, nylon mesh, glass mesh,porous plastic screen, PVDF, polystyrene, or other stabilizingstructure), porosity of materials, functional requirements (e.g., samplesize, reagent volumes, detection technology, time-to-result, incubation,heating, etc.), safety/handling requirements (e.g., self-containment,regulatory approval, ease of use, etc.), and/or the like, and in thecase of assays, assay requirements (e.g., binding assay, competitivebinding assay, single step assay, two-step assay, etc.).

The embodiment of the invention depicted in FIG. 15 for using theinvention in a sandwich ELISA assay, for example, involves the followingmaterials and methods for assembly. This embodiment uses the followingmaterials: a container (e.g. 96-well microtiter plate, injection moldedcommodity, etc.), hydrophobic porous structural material (e.g.polypropylene mesh, etc.), hydrophilic porous structural material (nylonmesh, etc.), paraffin wax with a melting temperature around 35° C.,mineral oil, primary antibody binding buffer (containing bufferingcomponents, salt components, detergent, protein components, etc.),paramagnetic particles conjugated to an antibody against a target,secondary conjugate antibody binding buffer (containing, a secondaryantibody conjugated to an enzyme, e.g. HRP, alkaline phosphatase, etc.),buffering components, salt components, detergent, protein components,etc.), and a substrate solution (e.g. TMB, para-Nitrophenylphosphate,etc.). Assembly and establishment of the stabilized layers and otherassay components is as follows: substrate solution is first added tobottom (surface) of the container. The container is then heated to above35° C. and liquid paraffin wax is added. Hydrophobic porous structuralmaterial cut to the appropriate dimensions (e.g. diameter, thickness,etc.), such that the material is press-fit into place once inside thecontainer, is added to the liquid paraffin. The container is thenbrought to room temperature (e.g. 22° C.) solidifying the paraffin intowax. A hydrophilic porous structural material (e.g. nylon), cut to theappropriate dimensions (e.g. diameter, thickness, etc.) is firstsubmerged in secondary conjugate antibody binding buffer, and thenplaced in the container. Mineral oil is added to the container, alongwith a hydrophobic porous structural material, cut to the appropriatedimensions (e.g. diameter, thickness, etc.). Lastly, primary antibodybinding buffer and paramagnetic particles conjugated to an antibodyagainst a target are then added to the container along with ahydrophilic porous structural material (e.g. nylon), cut to theappropriate dimensions (e.g. diameter, thickness, etc.).

In other embodiments, the assay is a RT-LAMP assay, for example. In someembodiments, reagents for RT-LAMP are dried or lyophilized onto thebottom surface of the container. In some embodiments, the stabilizedphases are established by first submerging an appropriate permeablematerial (based on, e.g. contact angle, pore size, porosity, etc.) intoa desired fluid and then placed in a container. In some embodiments, thelayers are assembled in a dry format whereby an appropriate porousmaterial for association with a phase or layer is submerged into adesired fluid, removed from said fluid, and then frozen (e.g. waxsolidification, water freezing, etc.). These components are then addedto the container in layers. In some embodiments an excess of fluid isadded to a container, and porous structural material, with and withoutan associated fluid, is added to the fluid. In some embodiments, theporous structural material is first placed in a container and fluid isadded. In some embodiments the temperature is changed to adjust thefluid phase to aid assembly. In some embodiments ambient pressure ischanged to adjust the fluid phase to aid assembly. In some embodiments,atmospheric gas composition is adjusted to aid assembly. In someembodiments, reagents are dried or lyophilized in the container. In someembodiments, solid components (e.g. salt crystals, PMPs, etc.) are addedto a stabilized phase before additional stabilized phases are layered ontop. In some embodiments, the assembly of stabilized phases are done inan automated fashion.

In some embodiments, a device or system of the invention, including adisposable and/or point-of-care device or assay or cartridge, is fittedwith bluetooth functionality (e.g., a chip with bluetooth radio) toallow transmission of results to a bluetooth-equipped device (e.g., aphone or computer). In some embodiments, system or device results orresults from a method as described herein are transmitted via bluetoothor other communication functionality (e.g. Wifi, near fieldcommunication, cellular networks, etc.) to another device (e.g. a phone,a tablet, a CPU, a computer, an imaging device, a storage device, etc.).

In some embodiments, a computer system is programmed or otherwiseconfigured to implement methods of the present disclosure (or isassociated with or comprises a device or system of the invention). Insome embodiments, a CPU or computer can execute a sequence ofmachine-readable instructions, which can be embodied in a program orsoftware. The instructions may be stored in a memory location. Theinstructions can be directed to the CPU, which can subsequently programor otherwise configure the CPU to implement methods of the presentdisclosure. Examples of operations performed by the CPU can includesample addition, addition of PMPs (or other target-binding solid phasematerial(s)), movement of a stabilizing interface structure, applicationof a magnetic or other force to a device or system (or in a method),heating, cooling or thermocycling. The CPU can be part of a circuit,such as an integrated circuit. One or more other components of thesystem can be included in the circuit. In some cases, the circuit is anapplication specific integrated circuit.

The computer system can also include a memory or memory location (e.g.,random-access memory, read-only memory, flash memory), electronicstorage unit (e.g., hard disk), communication interface forcommunicating with one or more other systems, and peripheral devices,such as cache, other memory, data storage and/or electronic displayadapters. The storage unit can be a data storage unit (or datarepository) for storing data. The computer system can be operativelycoupled to a computer network with the aid of the communicationinterface. The network can be the Internet, an internet and/or extranet,or an intranet and/or extranet that is in communication with theInternet. The network can include one or more computer servers, whichcan enable distributed computing, such as cloud computing. The network,in some cases with the aid of the computer system, can implement apeer-to-peer network, which may enable devices coupled to the computersystem to behave as a client or a server.

The foregoing description of illustrative embodiments of the disclosurehas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the disclosure to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of thedisclosure. The embodiments were chosen and described in order toexplain the principles of the disclosure and as practical applicationsof the disclosure to enable one skilled in the art to utilize thedisclosure in various embodiments and with various modifications assuited to the particular use contemplated. It is intended that the scopeof the disclosure be defined by the claims appended hereto and theirequivalents.

EXAMPLES Example 1: System and Method for LAMP-Based Detection ofSARS-CoV-2 RNA

The system and device and methods described in this Example comprisesreagents for LAMP-based detection of the target housed on a bottomsurface of the container. The system comprises two porous materials, alysis buffer, and a wash buffer. As shown in the FIG. 1, the system inthis embodiment is configured in layers in the following order, from topto bottom: (1) coconut oil, (2) lysis buffer with a glass mesh, (3)coconut oil with a porex mesh, (4) wash buffer with a glass mesh, (5)coconut oil with a porex mesh, and (6) reagents for LAMP reaction with aglass mesh.

Each porous material may be a hydrophilic glass mesh. Alternatively, oneporous material may be a glass mesh and the other porous material may bea synthetic hydrophobic polymer mesh.

In some embodiments, the biological sample is mixed with PMPs, forexample, and subsequently added to the container. In some embodiments,as shown in FIG. 12, the sample, saliva for example, can be addeddirectly to the lysis/binding buffer with PMPs which are already presentin the container of the invention. In some embodiments, like those shownin FIG. 12 and FIG. 13, coconut oil is replaced with a solidified wax.In these embodiments, the system in this embodiment is configured inlayers in the following order, from top to bottom: (1) lysis/bindingbuffer with PMPs, (2) solidified wax associated with polypropylenemeshes, (3) spatially multiplexed RT-LAMP reagents. In some embodiments,like those in FIG. 12 and FIG. 13, the system needs to be heated toabove the melting temperature of the wax before next steps are taken. Amagnet is applied to the bottom of the container, thereby drawing thetarget-PMP, SARS-CoV-2 RNA in this case, complexes through the layersand into contact with the LAMP reagents. In some embodiments, like thosein FIG. 13 and FIG. 14, a magnet is applied to the side of thecontainer, thereby drawing the target-PMP complexes through the layersand into contact with the LAMP reagents. The container may be incubatedat a suitable temperature (e.g. 65° C.) to perform the LAMP assay andsubsequently measure the resulting signal. In this embodiment, theresulting signal is colorimetric.

Example 2: System and Device with Layered Aqueous and Oil Phases

A side view of the device of the invention is shown in FIG. 2A. Thecontainer comprises a multi-well plate. One well contains stacked porousmaterials associated with oil (yellow). In the figure, the porousmaterials are synthetic hydrophobic polypropylene polymer mesh, referredto as in the Figure as “porex”, and hydrophilic glass mesh. The systemcontains 7 mesh materials. From top to bottom, the layers are asfollows: (1) lysis buffer (blue aqueous layer)(stabilized by glassmesh), (2) polypropylene polymer mesh (porex) (which stabilizes the oilphase), (3) wash buffer (red aqueous layer)(stabilized by glass mesh),(4) polypropylene polymer mesh (porex), (5) wash buffer (blue aqueouslayer)(stabilized by glass mesh), (6) polypropylene polymer mesh(porex), (7) LAMP reagents (red aqueous layer)(stabilized by glassmesh).

Example 3: Devices with Different Solid Substrates and Layers

FIG. 2B shows a bottom view and a top view of the system described inExample 1 above and shown in FIG. 1 following application ofparamagnetic particles and magnetic pull down. A comparison of a systemcomprising the synthetic polymer mesh, the synthetic polypropylenepolymer mesh and a glass mesh, and no porous material (e.g. only aqueousor oil layers) is shown. As shown in the FIG. 2B, the synthetic polymermesh (e.g. porex pad) and the combination of the porex pad and glassmesh both permit paramagnetic particles to pass through the porousmaterial.

Example 4: Modified Methods and Systems

To test whether additional modifications to the porous materials may bemade to further enhance bead pull down, small holes were created inglass mesh material and used in a system as described herein. FIG. 3shows a bottom view and a top view following magnetic bead pull downwhen these small holes were created in the glass mesh material. As shownin the figure, 1 mm holes allowed for significantly faster pulldown andlarger clumps of beads, and 0.5 mm holes allowed for faster pulldown.Accordingly, additional pores may be generated in the porous materialsto facilitate isolation of target-PMP complexes as needed.

Example 5: Solid Substrate Variations

Another embodiment of a system as described herein is shown in FIG. 4.As in FIG. 1, the system comprises reagents for LAMP-based detection ofthe target housed on a bottom surface (layer) of the container. In thisembodiment, the system comprises a Polytetrafluoroethylene (PTFE) O-ringto hold the LAMP reagents on the bottom of the container, and to providea firm surface for the porous material to rest on. In this embodiment,the porous material comprises polypropylene (PP) mesh. The systemcomprises a plurality of porous materials. In this instance, two porousmaterials are shown (e.g. two layers). Each porous material may comprisePP mesh. Alternatively, one material may comprise PP mesh and the othermaterial may comprise glass mesh. The system comprises a viral lysisbuffer and a wash buffer.

As shown in FIG. 4, the system is configured in layers in the followingorder, from top to bottom: (1) mineral oil, (2) viral lysis/RNA bindingbuffer (glass fabric/mesh), (3) mineral oil (PP mesh), (4) wash buffer(glass fabric/mesh), (5) mineral oil (PP mesh), (6) PTFE O-ring andreagents for LAMP reaction.

In this embodiment, a biological sample, for example, is mixed withPMPs, and subsequently added to the container. A magnet is applied tothe bottom of the container, thereby drawing the target-PMP complexesthrough the layers and into contact with the LAMP reagents. Thecontainer may be incubated at a suitable temperature (e.g. 65° C.) toperform the LAMP assay and subsequently measure the resulting signal. Inthis embodiment, the resulting signal is colorimetric.

Example 6: System and Device with Constrained LAMP Reagents

An image of a system as described in FIG. 4 containing a PTFE O-Ringholding the LAMP reagents on the bottom surface of the container isshown in FIG. 5. The image is shown after bead pull down, demonstratingthat the SARS-CoV-2 RNA target-PMP complexes are pulled down into thecenter of the O-Ring, thereby contacting the LAMP reagents. Red is shownto indicate where LAMP reagents are contained.

Example 7: System to Evaluate Hydrophilic Solid Substrate

Another embodiment of a system as described herein is shown in FIG. 6.In this embodiment, the system comprises a CNC-milled glycol modifiedPolyethylene Terephthalate (PETG) insert (stabilizing hydrophilicstructure) to hold the LAMP reagents on the bottom of the container.This custom PETG insert was manufactured to fit the exact geometry ofthe 96-well plate wells as seen in FIG. 7. The system comprises aplurality of porous materials. In this instance, two porous materialsare shown (e.g. two layers). Each porous material may comprise PP mesh.Alternatively, one material may comprise PP mesh and the other materialmay comprise a hydrophilic nylon mesh. Mesh porosity will generally besuch that target-PMP complexes can freely and robustly pass through fromone side to the other. The system comprises a lysis buffer and a washbuffer.

As shown in the figure, the system is configured in layers in thefollowing order, from top to bottom: (1) mineral oil, (2) lysis buffer(stabilized by nylon mesh), (3) mineral oil (stabilized by PP mesh), (4)wash buffer (stabilized by nylon mesh), (5) mineral oil (stabilized withPP mesh), and (6) reagents for LAMP reaction held within the PETGinsert.

The biological sample is mixed with PMPs, and subsequently added to thecontainer. A magnet is applied to the bottom of the container, therebydrawing the target-PMP complexes through the layers and into contactwith the LAMP reagents. The container may be incubated at a suitabletemperature (e.g. 65° C.) to perform the LAMP assay and subsequentlymeasure the resulting signal. In this embodiment, the resulting signalis colorimetric.

Example 8: System and Device with PETG Insert

In this Example, a colorimetric LAMP assay following magnetic bead pulldown using a system as described herein was carried out as seen in FIG.7. Colorimetric RT-LAMP reaction reagents were pipetted into wells offlat-bottomed 96-well polypropylene plate containing mineral oil. Abiological sample was mixed with PMPs and added to the system (referredto as a “bead-delivered template”). In the “Control” wells, no PETGinsert was present, and in the experimental wells (e.g. “+PETG Insert”,“Bead Delivered Template”) a PETG washer was present. The reactionscontained DNA primers, either a “New Primers” set or an “Old Primers”set, targeting DNA sequences specific to SARS-CoV-2. In the “Phase JumpMechanism . . . (2× water washes)” condition wells, from top to bottom,the wells contained mineral oil+Porex mesh, water+glass mesh layer,another mineral oil+Porex mesh layer, another water+glass mesh layer, athird oil+Porex mesh layer, and finally the PETG washer+LAMP reaction.

For the “Controls” and “+PETG Insert” conditions, SARS-CoV-2 template orwater was pipetted directly into the “+” or “−” wells respectively. Forthe “Phase Jump Mechanism . . . (2× water washes)” conditions, abiological sample containing either SARS-CoV-2 template (“+”) or water(“−”), was mixed with PMPs and added to the system (referred to as a“bead-delivered template”).

Data is compared to controls, and controls plus a PETG insert. As shownin FIG. 7, the system allowed for pull down of sufficient target-PMPcomplexes (visible as brown spots in the wells), and for successfulcolorimetric LAMP-based detection of the target. A positive reaction, orone in which SARS-CoV-2 DNA is detected, will turn yellow, whereas anegative reaction will remain pink. As shown, the system allowed forpull down of sufficient target-PMP complexes (visible as brown spots inthe wells), and for successful colorimetric LAMP-based detection of thetarget.

Example 9: System and Method with SARS-CoV-2 Limit of Detection

The results of a colorimetric LAMP assay to determine the limit ofdetection (LOD) using primers for SARS-CoV-2 with different numbers of“wash layers” are shown in FIG. 8. In all of the wells, colorimetricRT-LAMP reagents, containing DNA primers for SARS-CoV-2, were pipettedinto either PETG inserts or just the well itself (“Pre-Concentrated”wells) along with mineral oil. SARS-CoV-2 DNA was pre-concentrated andadded directly to the LAMP reactions in the “Pre-Concentrated” wells atthe number of copies listed on the left side of the figure. The“Concentrated” condition wells consisted of systems involving either 3wash layers (Top-Bottom: mineral oil+Porex mesh layer, water+glass meshlayer, a second mineral oil+Porex mesh layer, a second water+glass meshlayer, a third oil+Porex mesh layer, a third water+glass mesh layer, afourth mineral oil+Porex mesh layer, and finally the PETG+RT-LAMPreagents), or two wash layers (Top-Bottom: mineral oil+Porex mesh layer,water+glass mesh layer, a second mineral oil+Porex mesh layer, a secondwater+glass mesh layer, a third oil+Porex mesh layer, and finally thePETG+RT-LAMP reagents). To these wells, SARS-CoV-2 DNA, at the amountslisted on the left side of the figure, was added to saliva samples andlysis/binding buffer with PMPs, and then this mixture was added to thetops of the wells. After magnetic pulldown of the PMPs into contact withthe LAMP reactions, the 96-well plate was placed in an oven at 65° C.and taken out for imaging at 0 minutes, 20 minutes, and 35 minutes.

Example 10: System and Device with Saliva Sample

In this Example, a device and system of the invention was used tosuccessfully isolate target-PMP complexes from a saliva sample. From topto bottom, the systems comprised either a water+nylon mesh layer on topof a mineral oil+polypropylene mesh layer, or just a mineraloil+polypropylene mesh layer, on top of a PETG insert filled with water.The saliva sample was diluted with phosphate-buffered saline (PBS) andlysis buffer was added directly to the sample. The sample was heated to55° C. for 15 minutes, followed by heating to 98° C. for 3 minutes toheat-inactivate infectious material. The sample was cooled to roomtemperature, mixed with PMPs, and added to the container (e.g. added tothe wells of a multi-well plate containing the porous materials and washbuffer). A magnet was applied to the bottom of the container to draw thetarget-PMP complexes through the purification layers. As shown in thesystem, magnet size and thus field strength is critical to achievingadequate bead-pulldown. Additionally, this figure shows that undilutedsaliva performs better than diluted saliva in terms of overallbead-pulldown. The results are shown in FIG. 9.

Example 11: System and Device for 96-Well Container

In this Example, a container is prepared containing reagents forLAMP-based detection of a desired target housed on a bottom surface ofthe container. The reagents may be secured on the bottom surface by asuitable means, including an insert (e.g. PETG insert) or an O-Ring. Thesystem comprises a wash buffer and a plurality of porous materialsstacked within the container. The system comprises, from top to bottom:(1) polypropylene mesh associated with mineral oil, (2) wash bufferassociated with nylon mesh, (3) polypropylene mesh associated withmineral oil, (4) wash buffer associated with nylon mesh, (5)polypropylene mesh associated with mineral oil, and (6) a PETG insertwith LAMP reagents. The container may be pre-packed into a multi-wellplate, wherein each well of the plate contains the contents of a singlecontainer. This multi-well plate may be packaged into a kit.

The system further comprises a magnet. In this case, the magnet is anarray such that each magnet in the array can be aligned with a singlewell in the multi-well plate. For example, a biological sample is lysed,mixed with paramagnetic particles, added to the multi-well plate, andthe magnetic array is placed in a suitable position proximal to thebottom of the plate to draw the target-PMP complexes through thepurification layers (e.g. through the porous materials and the washbuffer) and into contact with the LAMP reagents. The plate is incubatedat 65° C., and a signal (e.g. colorimetric or fluorescent signal) ismeasured. FIG. 10 is a schematic showing an exemplary overview of thisembodiment.

Example 12 System and Device with Insert

Another overview of a system of the invention, in this case, forisolation and detection of analytes. FIG. 11A shows paramagneticparticles in the aqueous phase (i). Application of the magnetic forcebelow the system pulls the paramagnetic particles (e.g. the target-PMPcomplexes) through the oil phase (ii, iii, and iv) towards the bottomsurface of the system.

FIG. 11B shows an exemplary container holding the system. The bottomsurface of the container contains reagents for detection of the analyte(shown in red). The aqueous and oil phases are stabilized by maximizing,or otherwise optimizing fluid retentive forces, via solid substrateadhesion and surface tension, compared to buoyancy forces.

FIG. 11C shows an exemplary process for isolating and detecting ananalyte using a system as described herein, using an insert to house thesystem.

Example 13: LAMP-Based Detection of a Target from a Complex BiologicalSample

This system is designed for use in LAMP-based detection of a target froma complex biological sample. The system comprises a plurality of porousmaterials with different surface properties and reagents for LAMP-based,or RT-LAMP-based detection of a target housed on a bottom surface of thecontainer. The system also comprises a lysis/binding buffer and cancontain a wash buffer.

When LAMP reagents are in liquid form, and depending on the biologicalsample, the system may be configured in layers in the following order,from top to bottom: (1) mineral oil (optional), (2) lysis/bindingbuffer, (3) hydrophobic porous material (and associated mineral oil),and (4) reagents for LAMP (RT-LAMP) reaction. In other embodiments, theorder of layers 1-3 is optional, and may be rearranged as desired.

In some embodiments, mineral oil is replaced with solidified wax whichmelts at operational temperatures.

If the inclusion of an aqueous wash layer is deemed beneficial ornecessary, the system is then configured in layers in the followingorder, from top to bottom: (1) mineral oil (optional), (2) lysis/bindingbuffer, (3) hydrophobic porous material (and associated mineral oil),(4) hydrophilic porous material (wash buffer), (5) another hydrophobicporous material (and associated mineral oil), and (6) reagents for LAMPreaction. In other embodiments, the order of layers 1-4 is optional, andmay be rearranged as desired.

When dry versions of LAMP reagents are used instead of liquid, thesystem layers are configured in the following order: (1) mineral oil(optional), (2) lysis/binding buffer, (3) hydrophobic porous material(and associated mineral oil), (4) an aqueous reconstitution buffer,mineral oil, and finally (5) the dried LAMP reagents. In this lastconfiguration with dry LAMP reagents, oil/wax must be able to besolidified to keep the reconstitution buffer and dry lamp reagentsseparate until the device is heated, allowing the aqueous reconstitutionbuffer to combine with the dry reagents to then mimic the previousconfiguration where wet LAMP reagents are used. In other embodiments,the order of layers 1-4 is optional, and may be rearranged as desired.

Each porous material may be a hydrophobic mesh (polypropylene or othersynthetic or natural polymer). Alternatively, one porous material may bea glass mesh and the other porous material may be a synthetic polymermesh. The biological sample may be mixed with PMPs, for example, andsubsequently added to the container, or the PMPs are already in thebinding buffer and the biological sample can be simply added and mixeddirectly into the container. In some embodiments, the lysis ofbiological components contained in a complex biological sample iscarried out using ultrasonication. In some embodiments, a sonotrode usedto provide the ultrasonic vibrations, is applied externally to thedevice body. In some embodiments, the sonotrode, or similar device, isintegrated into the body of a target-positioning device of theinvention. A magnet is applied to the bottom of the container, therebydrawing the target-PMP complexes through the layers and into contactwith the LAMP reagents.

In some embodiments, the portion of the device body in contact with theLAMP reagents is divided into more than one compartment to facilitatespatially-multiplexed reactions. The container may be incubated at asuitable temperature (e.g. 65° C.) to perform the LAMP assay andsubsequently measure the resulting signal. In this embodiment, theresulting signal can be colorimetric, turbidimetric, or fluorometric,for example.

Example 14: RT-qPCR-Based Detection of a Target from a ComplexBiological Sample

In this Example, a system of the invention is designed for use in theRT-qPCR-based detection of a target from a complex biological sample.This system comprises reagents for PCR-based, or RT-PCR-based detectionof the target housed at a bottom layer of the system or on a bottomsurface of the container. The container housing the PCR reaction can bea simple cup shape at the bottom of the device, or in some embodiments,the geometry of the container housing the reaction can be such thatthermocycling and is more efficient. For example, in some embodiments,the container housing the reaction can have a high aspect ratio tofacilitate quicker transfer of heat (i.e., reducing the distance overwhich temperature must be conducted to facilitate temperature cycling ofthe reaction). In some embodiments, the container housing the reactioncomprises, consists essentially of, or consists of a microfluidicchannel. In some embodiments, the container housing the reaction is madeof different material as the rest of the device. In some embodiments,the container housing the reaction has integrated heating elements init. The system can comprise a plurality of porous materials withdifferent surface properties. The system comprises a lysis/bindingbuffer and can contain a wash buffer. Potential layer configurationsmatch those of LAMP with the exception that RT-qPCR reagents would beused in place of LAMP reagents (either in liquid form or dry). UnlikeLAMP/RT-LAMP which is an isothermal reaction, the container with RT-qPCRreagents may be thermocycled at appropriate temperatures (e.g. 95° C.for 10 s, 60° C. for 30 s) to perform the PCR (RT-PCR) assay andsubsequently measure the resulting signal. In this embodiment, theresulting signal is fluorometric.

Example 15: Target Isolation from a Complex Biological Sample for NextGeneration Sequencing

In this Example, a system of the invention is designed for isolation ofa target from a complex biological sample for sequencing. This systemcomprises reagents for next-generation sequencing (NGS) of the targethoused toward or on a bottom surface of the container. However, unlikeLAMP/RT-LAMP and RT-qPCR embodiments, this embodiment would be used forisolation, initial amplification reactions, and transfer of targetmaterial and may, but does not necessarily need, to include any endpointdetection. Instead, the isolated material can be used for nearly anydownstream process.

In fact, the application of NGS is just one of many potentialapplications where isolated material may be used in a downstream processpotentially outside a device of the invention. Indeed, depending on theanalyte (e.g., cells, protein, nucleic acid, or glycoprotein) and theapplication (enumeration of cancer cells, isolation protein for massspectrometry, or detection on a lateral or vertical flow device) anenumerable number of potential downstream uses are enabled. Thus, NGS isonly used here as one relevant example. In the case of NGS, isolatednucleic acid is used with downstream equipment for sequencing. NGSsometimes requires isothermal, or may require thermocycling, forpreamplification of material prior to sequencing. Therefore, a system,device or method of the invention may be used to pre-amplify theisolated material prior to transfer of the material into sequencingequipment. Pre-amplification can also be performed outside of a deviceas described after initial isolation and transfer.

In the case of NGS, in some embodiments, the container housing thereagents for stabilizing or buffering the isolated analyte for NGS isdetachable. In some embodiments, the container housing the reagents forNGS facilitates transfer of NGS reagents to a new container. As withprevious descriptions of layer configurations, NGS reagents may be inliquid form or dried and reconstituted with a layer of reconstitutionbuffer. In some embodiments, the NGS reagents are adhered to the surfaceof a detachable element in the bottom of the container. In someembodiments, devices of the invention are arrayed to interface withmicrotiter plates (48-well, 96-well, 384-well) such that the magnet isable to pull the analyte into the microtiter plate. In theseembodiments, the NGS reagents may be housed in the wells of themicrotiter plate, and the device of the invention, housing the othercomponents of the method of the invention, is placed into the top of thewell. In some embodiments the container housing the reagents for NGS isa simple cup, or in some embodiments, the geometry of the containerhousing the reaction can be such that thermocycling and is moreefficient. In some embodiments, the container housing the NGS reagentshas a high aspect ratio to facilitate quicker transfer of heat. In someembodiments, the container housing the reaction comprises, consistsessentially of, or consists of a microfluidic channel.

In some embodiments, the container housing the reaction is made ofdifferent material as the rest of the device. In some embodiments, thecontainer housing the reaction has integrated heating elements in it.The system comprises a plurality of porous materials with differentsurface properties. The system comprises a lysis/binding buffer and cancontain a wash buffer. Depending on the biological sample, the systemmay be configured in layers in the following order, for example, fromtop to bottom: (1) mineral oil, (2) lysis/binding buffer, (3)hydrophobic porous material (mineral oil) and (4) reagents for NGS. Ifthe inclusion of an aqueous wash layer is deemed beneficial ornecessary, the system may be configured in layers in the followingorder, for example, from top to bottom: (1) mineral oil, (2)lysis/binding buffer, (3) hydrophobic porous material (mineral oil), (4)hydrophilic porous material (wash buffer), (5) another hydrophobicporous material (mineral oil), and (6) reagents for NGS. Each porousmaterial may comprise a hydrophobic mesh (e.g. polypropylene or othersynthetic or natural polymer). Alternatively, one porous material maycomprise a glass mesh and the other porous material may comprise asynthetic polymer mesh.

The biological sample may be mixed with PMPs, and subsequently added tothe container, or the PMPs are already in the binding buffer and thebiological sample can be simply added and mixed directly into thecontainer. A magnet is applied to the bottom of the container, therebydrawing the target-PMP complexes through the layers and into contactwith the NGS (RT-PCR) reagents. The container may be thermocycled atappropriate temperatures (e.g. 95° C. for 10 s, 60° C. for 30 s) toperform the initial steps of NGS.

Example 16: Sandwich ELISA

In some embodiments of the invention, stabilized interfaces are used topartition and stabilize the reagents used to run a sandwich ELISA on abiological sample including a primary antibody binding buffer, aconjugated secondary antibody binding buffer, and a substrate solution,with mineral oil and solidified wax layers (MT=35° C.) separating eachlayer. See FIG. 15.

In this assay embodiment, a biological sample is added to allow thesample to mix with the topmost layer within the device which containsprimary antibody binding buffer composed of a buffer, paramagneticparticles (PMPs) conjugated to a primary capture antibody(target-specific), and other various components. After a period ofincubation, a magnetic force is applied perpendicular to the porousmeshes which pulls the beads through the mineral oil layer and into thesecondary antibody binding buffer composed of a buffer, secondaryconjugate antibodies (HRP or other enzyme), and salt and bufferingcomponents which establish optimal antibody-target binding other variouscomponents. Due to the presence of a solid wax layer beneath this layer,the beads stop in the aqueous layer. In the secondary antibody bindingbuffer, enzyme-conjugated antibodies are allowed to bind other regionson the target, which is already bound by the primary antibody/PMP. Afteran incubation period, temperature can be increased to above the meltingtemperature of the wax layer, which allows the beads to be pulledthrough (i.e. another ESP wash), and into the substrate solution. Inthis layer, enzyme substrate is converted into a fluorescent orcolorimetric product which can be measured using traditional means.

In another format of this assay, the top layer of the device containsboth PMPs that can bind the target as well as primary antibodies labeledwith HRP or other substrate converting enzyme. Then the target isisolated into a final buffer containing a substrate solution. The enzymeconverts the substrate into a fluorescent or colorimetric product fordetection via traditional means.

Example 17: CTC Capture

In another embodiment of the invention, stabilized interfaces are usedto partition and stabilize the reagents needed to isolate circulatingtumor cells (CTCs) from a blood-based sample (peripheral bloodmononuclear cells (PBMCs)) including CTC binding Buffer, abuffer-labeling reagents to differentiate CTCs from non-CTCs, and afinal aqueous volume, all separated by either mineral oil or a solid waxwith a melting temperature of ˜35° C. See FIG. 16.

In this assay, a blood-based sample, either whole blood or PBMCs, isadded to the top of a well containing one or porous meshes. Once added,the sample mixes with the topmost layer which contains CTC bindingbuffer composed of a phosphate-buffered saline (PBS), paramagneticparticles (PMPs) conjugated to a capture antibody (CTC-specifictargets), and other various components such as salts and buffer whichhelp preserve CTC viability and to help establish optimal bindingconditions. After a period of incubation, a magnetic force is appliedperpendicular to the porous meshes which pulls the beads through themineral oil layer and into a fluorescent antibody binding buffer. Due tothe presence of a solid wax layer beneath this layer, the beads stop inthe aqueous layer. In the fluorescent antibody binding buffer,fluorescently-tagged antibodies are allowed to bind targets on thecaptured CTCs. After an incubation period, temperature can be increasedto above the melting temperature of the wax layer, which allows thebeads to be pulled through (i.e. another ESP wash), and into an aqueousphase (PBS). In this layer, CTCs can be counted via fluorescentmicroscopy.

Example 18: Inputs for Other Methods and Devices

The ability to quickly go from raw sample to purified sample is abottleneck in most assay platforms due to complexity and cost. Theinventions described herein provide a simple solution to isolate targetsfrom raw samples (e.g., blood, urine, saliva, plasma) directly into afinal destination such as the input of another device or method. In someembodiments, this may be a microtiter plate. In other embodiments, adevice of the invention can form the sample loading chamber for amicrofluidic device. After isolation and transfer of the target into themicrofluidic device chamber, microfluidic controls can be used tosubsequently process the sample as needed.

In another embodiment, just as PMPs can be transferred into a microwellplate, the PMPs can be transferred onto a lateral flow assay (LFA) orsimilar assay (e.g., vertical flow assay) where analyte can then bedetected. For example the PMPs can be transferred to the sample pad ofan LFA. A low pH elution could be applied to the LFA to elute theanalyte bound via IgG to the PMPs. The pore size of the sample padmaterial can prevent migration of the PMPs down the LFA or may befiltered at a subsequent junction with another material of smaller poresize. Eluted material can then flow onto a next pad laden with driedneutralization buffer to renormalize the pH. The eluate can thenrehydrate pad material laden with conjugate or biotinylated antibody,allowing the conjugate/Ab to bind the eluted target and flow downstreamfor detection. Downstream detection can be performed in a variety ofways known in the area of flow assay design. Similarly, a device, systemor method of the invention can be used to isolate target into analogousvertical flow assays.

Example 19: Isolation of Nucleic Acid for Stable Transport

Devices, systems and methods of the invention can also be used toisolate target onto an LFA sample pad, they can also be used to isolatetarget onto a pad for drying for stable storage and transport. Unlike adried blood spot which would contain background contaminants from asmall volume of blood (μL's), a target isolated using a device, systemor method of the invention would represent the isolated target from amuch larger volume of blood (mL's) and most contaminating substanceswill have been removed.

What is claimed is:
 1. A system for isolating a target from a sample, the system comprising at least one aqueous phase and at least one oil phase stabilized in proximity to one another within a container by inclusion of one or more porous structural materials associated with the aqueous phase or the oil phase or both.
 2. The system of claim 1, wherein the at least one aqueous phase or the at least one oil phase or both are stabilized within the container by a hydrophilic porous material associated with the at least one aqueous phase and/or a hydrophobic porous material associated with the at least one oil phase.
 3. The system of claim 1 or 2, wherein the at least one aqueous phase and the at least one oil phase are stabilized within the container by modulating geometry or one or more chemical or physical material characteristics selected from density, surface chemistry, and porosity of a hydrophilic/hydrophobic porous material or a material having a preference for a phase or layer present in the system.
 4. The system of claim 3, wherein the at least one aqueous phase and the at least one oil phase are stabilized within the container by a hydrophilic porous material associated with the at least one aqueous phase, a hydrophobic porous material associated with the at least one oil phase, and by modulating surface chemistry such that the buoyancy forces of either the at least one oil phase and/or the at least one aqueous phase is less than the surface tension forces.
 5. The system of claim 1, wherein the system comprises a first aqueous phase, a second aqueous phase, a first oil phase, and a second oil phase.
 6. The system of claim 5, wherein the phases are stacked in an alternating fashion within the container, such that the first and second aqueous phase are not in direct contact with one another and the first and the second oil phases are not in direct contact with one another.
 7. The system of claim 1, wherein the container comprises a top opening to permit addition of a sample to the container.
 8. The system of claim 7, wherein the at least one aqueous phase is closest to the top opening of the container.
 9. The system of claim 7, wherein the at least one oil phase is closest to the top opening of the container.
 10. The system of claim 1, wherein the at least one aqueous phase comprises a lysis buffer.
 11. The system of any claim 1, wherein the at least one aqueous phase comprises a wash buffer.
 12. The system of claim 1, further comprising paramagnetic particles (PMPs).
 13. The system of claim 12, wherein PMPs are housed within the container.
 14. The system of claim 12, wherein PMPs are functionalized.
 15. The system of claim 13, wherein PMPs are housed within the at least one aqueous phase.
 16. A system for moving or isolating a target analyte from a sample, the system comprising a first aqueous phase, a second aqueous phase, a first oil phase, and a second oil phase wherein: a. the phases are stacked in an alternating fashion within a container, such that the first and second aqueous phases are not in direct contact with one another and the first and the second oil phases are not in direct contact with one another, b. the phases are stabilized within the container by: i. a hydrophilic porous material associated with the first aqueous phase, ii. a hydrophilic porous material associated with the second aqueous phase; iii. a hydrophobic porous material associated with first oil phase; and iv. a hydrophobic porous material associated with the second oil phase.
 17. The system of claim 16, wherein the phases are further stabilized within the container by modulating surface chemistry such that the preferences of the hydrophobic and/or hydrophilic materials for the oil and aqueous phases, respectively, prevail over one or more other forces in the system to maintain system stability.
 18. The system of claim 16, wherein the container comprises a top opening to permit addition of a sample to the container.
 19. The system of claim 18, wherein the first aqueous phase is closest to the top opening of the container or the first oil phase is closest to the top opening of the container.
 20. The system of claim 17, wherein the container is an insert.
 21. The system of claim 16, wherein the first aqueous phase or the second aqueous phase or both comprises a lysis buffer.
 22. The system of claim 16, wherein the first aqueous phase or the second aqueous phase or both comprises a wash buffer.
 23. The system of claim 16, further comprising paramagnetic particles (PMPs).
 24. The system of claim 23, wherein PMPs are housed within the container.
 25. The system of claim 23 or 24, wherein the PMPs are functionalized.
 26. The system of claim 23, wherein PMPs are housed within the first aqueous phase.
 27. The system of claim 1, further comprising a magnet.
 28. The system of claim 16, further comprising a magnet.
 29. The system of claim 1, wherein the container comprises a multi-well plate.
 30. The system of claim 16, wherein the container comprises a multi-well plate.
 31. The system of claim 1, wherein the sample comprises a nasopharyngeal sample, an oropharyngeal sample, an oral swab sample, an oral sponge sample, a nasal swab sample, a mid-turbinate sample, a saliva sample, a blood sample, a urine sample, a stool sample, a cerebrospinal fluid sample, a sputum sample, a cheek swab, or a biopsy sample.
 32. The system of claim 1, wherein the target is selected from the group consisting of a nucleic acid, a viral nucleic acid and a SARS-CoV-2 nucleic acid.
 33. The system of claim 16, wherein the sample comprises a nasopharyngeal sample, an oropharyngeal sample, an oral swab sample, an oral sponge sample, a nasal swab sample, a mid-turbinate sample, a saliva sample, a blood sample, a urine sample, a stool sample, a cerebrospinal fluid sample, a sputum sample, a cheek swab, or a biopsy sampled.
 34. The system of claim 16, wherein the target is selected from the group consisting of a nucleic acid, a viral nucleic acid and a SARS-CoV-2 nucleic acid.
 35. The system of claim 1, further comprising reagents for detecting the target housed within the container, and wherein the reagents for detecting the target may comprise reagents for a loop mediated isothermal amplification (LAMP) or a reverse transcriptase loop mediated isothermal amplification (RT-LAMP) assay.
 36. The system of claim 16, further comprising reagents for detecting the target housed within the container, and wherein the reagents for detecting the target may comprise reagents for a loop mediated isothermal amplification (LAMP) or a reverse transcriptase loop mediated isothermal amplification (RT-LAMP) assay.
 37. The system of claim 35 or 36, wherein the LAMP or RT-LAMP assay is a colorimetric assay or a fluorescent assay.
 38. A method for determining the presence or amount of a target in a sample, the method comprising (a) adding a sample and target-binding paramagnetic particles to a system according to claim 1 or 16, (b) applying a magnetic force, and (d) determining the presence or amount of the target.
 39. A method for moving or isolating a target from a sample, the method comprising: a. adding a sample to a system comprising at least one aqueous phase and at least one oil phase stabilized in proximity to one another within a container; and b. applying a magnetic force to the system, wherein the sample is contacted with paramagnetic particles (PMPs) prior to applying the magnetic force to the system, wherein contacting the sample with the paramagnetic particles generates one or more target-PMP complexes, and wherein applying the magnetic force to the system draws the target-PMP complexes through the at least one aqueous phase and the at least one oil phase towards a bottom surface of the container.
 40. The method of claim 39, wherein the at least one aqueous phase and the at least one oil phase are stabilized within the container by a hydrophilic porous material associated with the at least one aqueous phase and/or a hydrophobic porous material associated with the at least one oil phase.
 41. The method of claim 39, wherein the at least one aqueous phase and the at least one oil phase are stabilized within the container by modulating geometry or one or more chemical or physical material characteristics selected from density, surface chemistry, and porosity of a hydrophilic/hydrophobic porous material or a material having a preference for a phase or layer present in the system.
 42. The method of claim 41, wherein the at least one aqueous phase and the at least one oil phase are stabilized within the container by a hydrophilic porous material associated with the at least one aqueous phase, a hydrophobic porous material associated with the at least one oil phase, and by modulating surface chemistry such that the preferences of the hydrophobic and/or hydrophilic materials for the oil and aqueous phases, respectively, prevail over one or more other forces in the system to maintain system stability.
 43. The method of claim 39, wherein the system comprises a first aqueous phase, a second aqueous phase, a first oil phase, and a second oil phase.
 44. The method of claim 43, wherein the phases are stacked in an alternating fashion within the container, such that the first and second aqueous phase are not in direct contact with one another and the first and the second oil phases are not in direct contact with one another.
 45. The method of claim 39, wherein the container comprises a top opening to permit addition of a sample to the container.
 46. The method of claim 45, wherein the at least one aqueous phase is closest to the top opening of the container.
 47. The method of claim 46, wherein the at least one oil phase is closest to the top opening of the container.
 48. The method of claim 39, wherein the at least one aqueous phase comprises a lysis buffer.
 49. The method of claim 39, wherein the at least one aqueous phase comprises a wash buffer.
 50. The method of claim 39, wherein the PMPs comprise functionalized PMPs.
 51. The method of claim 39, wherein PMPs are housed within the container.
 52. The method of claim 51, wherein PMPs are housed within the at least one aqueous phase.
 53. The method of claim 39, wherein the PMPs are contacted with the sample prior to adding the sample to the system.
 54. The method of claim 39, further comprising detecting the target in the sample.
 55. The method of claim 54, wherein the system further comprises reagents for detection of the target housed on within the container, and wherein detecting the target comprises drawing the target-PMP complexes through the plurality of porous materials and to the reagents for detection of the target.
 56. The method of claim 55, wherein the reagents comprise reagents for a loop mediated isothermal amplification (LAMP) or a reverse transcriptase loop mediated isothermal amplification (RT-LAMP) assay, and wherein detecting the target comprises detecting a signal generated during the LAMP or RT-LAMP assay.
 57. The method of claim 56, wherein the LAMP or RT-LAMP assay is a colorimetric assay or a fluorescent assay.
 58. A method of moving a target away from other materials in a sample, the method comprising a. adding a sample to a system comprising a first aqueous phase, a second aqueous phase, a first oil phase, and a second oil phase, wherein: the phases are stacked in an alternating fashion within the container, such that the first and second aqueous phases are not in direct contact with one another and the first and the second oil phases are not in direct contact with one another, and the phases are stabilized within the container by: i. a hydrophilic porous material associated with the first aqueous phase, ii. a hydrophilic porous material associated with the second aqueous phase; iii. a hydrophobic porous material associated with first oil phase; and iv. a hydrophobic porous material associated with the second oil phase; and b. applying a magnetic force to the system, wherein the sample is contacted with target-binding paramagnetic particles (PMPs) prior to applying the magnetic force to the system, wherein contacting the sample with the target-binding paramagnetic particles generates one or more target-PMP complexes, and wherein applying the magnetic force to the system draws the target-PMP complexes through the phases towards a bottom surface of the container.
 59. The method of claim 58, wherein the phases are further stabilized within the container by modulating surface chemistry such that the preferences of the hydrophobic and/or hydrophilic materials for the oil and aqueous phases, respectively, prevail over one or more other forces in the system to maintain system stability.
 60. The method of claim 58, wherein the container comprises a top opening to permit addition of a sample to the container.
 61. The method of claim 60, wherein the first aqueous phase is closest to the top opening of the container.
 62. The method of claim 60, wherein the first oil phase is closest to the top opening of the container.
 63. The method of claim 58, wherein the first aqueous phase or the second aqueous phase or both the first and second aqueous phase comprises a lysis buffer.
 64. The method of claim 58, wherein the first aqueous phase or the second aqueous phase or both the first and second aqueous phase comprises a wash buffer.
 65. The method of claim 58, wherein the target-binding PMPs are functionalized.
 66. The method of claim 58, wherein the target-binding PMPs are housed within the container.
 67. The method of claim 66, wherein target-binding PMPs are housed within the first aqueous phase or the second aqueous phase or both the first and second aqueous phase.
 68. The method of claim 66, further comprising negative- or positive-control PMPs or both.
 69. The method of claim 68, wherein the sample is a nasopharyngeal sample, an oropharyngeal sample, an oral swab sample, an oral sponge sample, a nasal swab sample, a mid-turbinate sample, a saliva sample, a blood sample, a urine sample, a stool sample, a cerebrospinal fluid sample, a sputum sample, a cheek swab, or a biopsy sample.
 70. The method of claim 68, wherein the sample is obtained from a subject suspected of having an infection.
 71. The method of claim 70, wherein the subject is suspected of having a viral infection.
 72. The method of claim 71, wherein the subject is suspected of having a viral upper respiratory infection.
 73. The method of claim 70, wherein the subject is suspected of having an infection selected from SARS-CoV2, SARS, a coronavirus, rhinovirus, influenza, and respiratory syncytial virus.
 74. The method of claim 58, wherein the target comprises viral nucleic acid.
 75. The method of claim 74, wherein the target comprises a SARS-CoV-2 nucleic acid.
 76. In a disposable cartridge comprising a flow-through assay to determine the presence or amount of a target in a sample of a fluid comprising a sample application space, a cartridge top, a cartridge bottom, reagents for detection or quantification of the target and an enclosure, the improvement comprising employing within the enclosure target-binding paramagnetic particles, at least one aqueous phase and at least one oil phase stabilized in proximity to one another within a container by inclusion of a porous structural material associated with the aqueous phase or the oil phase or both, and, optionally, a magnet.
 77. In a flow assay device comprising a sample application portion, a conjugate portion, a test portion and pre-immobilized reagents in different parts of the device, the improvement comprising employing target-binding paramagnetic particles, at least one aqueous phase and at least one gaseous or oil phase stabilized in proximity to one another by inclusion of a porous structural material associated with the aqueous phase or the gaseous or oil phase or both, and optionally a magnet.
 78. A flow assay device according to claim 77, wherein the flow assay is a lateral flow assay.
 79. A flow assay device according to claim 77, wherein the flow assay is a vertical flow assay.
 80. In an immunometric assay to determine the presence, concentration or amount of a target substance in a sample comprising forming a ternary complex of a first labeled binding agent, said target substance, and a second binding agent said second binding agent being bound to a solid carrier wherein the presence or amount of the substance in the sample is determined by measuring either the amount of labeled binding agent bound to the solid carrier or the amount of unreacted labeled binding agent, the improvement comprising employing target-binding paramagnetic particles, at least one aqueous phase and at least one oil phase stabilized in proximity to one another by inclusion of a porous structural material associated with the aqueous phase or the oil phase or both, and optionally a magnet.
 81. In a nucleic acid amplification test to determine the presence or amount of a target substance in a sample comprising amplifying a nucleic acid sequence and detection of the sequence, the improvement comprising employing target-binding paramagnetic particles, at least one aqueous phase and at least one oil phase stabilized in proximity to one another by inclusion of a porous structural material associated with the aqueous phase or the oil phase or both, and optionally a magnet.
 82. A nucleic acid amplification test according to claim 81, wherein the nucleic acid amplification test is PCR or RT-PCR.
 83. A nucleic acid amplification test according to claim 81, wherein the nucleic acid amplification test is isothermal.
 84. A nucleic acid amplification test according to claim 83, wherein the isothermal nucleic acid amplification test is reverse transcription polymerase chain reaction (RT-PCR), nicking endonuclease amplification reaction (NEAR), transcription mediated amplification (TMA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), clustered regularly interspaced short palindromic repeats (CRISPR), strand displacement amplification (SDA). 